Conformationally Specific Viral Immunogens

ABSTRACT

The present invention provides methods of making engineered viral proteins and protein complexes that are useful as vaccine immunogens, engineered viral proteins and protein complexes made using such methods, and pharmaceutical compositions comprising such engineered viral proteins and protein complexes. Such engineered viral proteins and protein complexes may comprise one or more cross-links that stabilize the conformation of an antibody epitope, such as a quaternary neutralizing antibody, and may exhibit an enhanced ability to elicit a protective immune response when administered to a subject as a component of a vaccine.

This application claims the benefit of U.S. Provisional PatentApplication No. 61/644,830, filed May 9, 2012, the contents of which arehereby incorporated by reference.

A portion of the disclosure of this patent document contains materialwhich is subject to copyright protection. The copyright owner has noobjection to the facsimile reproduction by anyone of the patent documentor the patent disclosure as it appears in the Patent and TrademarkOffice patent file or records, but otherwise reserves all copyrightrights whatsoever.

FIELD OF THE INVENTION

The present invention relates, in part, to methods of producingconformationally-specific immunogens, and methods of producingengineered viral proteins and protein complexes useful asconformationally-specific immunogens, and to conformationally-specificimmunogens and engineered viral proteins and protein complexes producedusing such methods.

BACKGROUND OF THE INVENTION

Many pathogenic viruses have developed strategies to evade recognitionand elimination by host immune systems. Such strategies include highmutation rates of envelope glycoproteins, glycosylation of envelopeproteins, and conformational masking—whereby conserved portions of viralproteins, such as those involved in key functions such as receptorbinding, are “masked” such that they are poorly recognized by, or evaderecognition by, antibodies. Such conformational masking poses a majorproblem in the development of vaccines based on viral proteins. Hencethere is a need in the art for methods of producing engineered viralproteins, and complexes of viral proteins, that have enhancedimmunogenicity and enhanced effectiveness as vaccines.

SUMMARY OF THE INVENTION

The present invention provides, in part, methods for producingconformationally-specific vaccine immunogens, methods of engineeringviral proteins and protein complexes, viral proteins and proteincomplexes so engineered, and pharmaceutical compositions comprising suchengineered viral proteins and protein complexes. Such engineeredproteins may be useful as conformationally-specific vaccine immunogens.

In one embodiment the present invention provides a method for producinga conformationally-specific immunogen, the method comprising: (a)obtaining a viral protein or protein complex in one or moreconformations that favor the elicitation of protective immune responses,(b) identifying one or more regions in the tertiary and/or quaternarystructure of the viral protein or protein complex in which theintroduction of one or more cross-links could stabilize the conformationof an antibody epitope (and/or could stabilize a conformation thatfavors the elicitation of a protective immune response), and (c)introducing into the viral protein or protein complex one or moretargeted cross-links at one or more of the regions identified in step(b) to form an engineered viral protein or protein complex, wherein theengineered viral protein or protein complex has one or more of thefollowing properties: (i) enhanced ability bind to a neutralizingantibody as compared to the viral protein or protein complex (i.e. ascompared to the viral protein or protein complex without or beforeintroduction of the cross-links), (ii) enhanced ability bind to abroadly neutralizing antibody as compared to the viral protein orprotein complex, (iii) enhanced ability bind to and activate B cellreceptors as compared to the viral protein or protein complex, (iv)enhanced ability to elicit an antibody response in an animal as comparedto the viral protein or protein complex, (v) enhanced ability to elicita protective antibody response in an animal as compared to the viralprotein or protein complex, (vi) enhanced ability to elicit productionof neutralizing antibodies in an animal as compared to the viral proteinor protein complex, (vii) enhanced ability to elicit production ofbroadly neutralizing antibodies in an animal as compared to the viralprotein or protein complex, (viii) enhanced ability to elicit aprotective immune response in an animal as compared to the viral proteinor protein complex, and (ix) enhanced ability to bind to and elicitproduction of antibodies that recognize quaternary neutralizing epitopesin an animal as compared to the viral protein or protein complex. Insome such embodiments the targeted cross-links are dityrosine (DT)cross-links.

In another embodiment the present invention provides a method forproducing a conformationally-specific immunogen, the method comprising:(a) obtaining a viral protein or protein complex in one or moreconformations that favor the elicitation of protective immune responses,and (b) introducing into the viral protein or protein complex one ormore cross-links that are stable under physiological conditions, whereinthe engineered viral protein or protein complex has one or more of thefollowing properties: (i) enhanced ability bind to a neutralizingantibody as compared to the viral protein or protein complex (i.e. ascompared to the viral protein or protein complex without or beforeintroduction of the cross-links), (ii) enhanced ability bind to abroadly neutralizing antibody as compared to the viral protein orprotein complex, (iii) enhanced ability bind to and activate B cellreceptors as compared to the viral protein or protein complex, (iv)enhanced ability to elicit an antibody response in an animal as comparedto the viral protein or protein complex, (v) enhanced ability to elicita protective antibody response in an animal as compared to the viralprotein or protein complex, (vi) enhanced ability to elicit productionof neutralizing antibodies in an animal as compared to the viral proteinor protein complex, (vii) enhanced ability to elicit production ofbroadly neutralizing antibodies in an animal as compared to the viralprotein or protein complex, (viii) enhanced ability to elicit aprotective immune response in an animal as compared to the viral proteinor protein complex, and (ix) enhanced ability to bind to and elicitproduction of antibodies that recognize quaternary neutralizing epitopesin an animal as compared to the viral protein or protein complex. Insome such embodiments the cross-links are targeted to identified and/orselected positions within the protein or protein complex's tertiary orquaternary structure. In some such embodiments the targeted cross-linkscomprise dityrosine (DT) cross links.

In some embodiments where DT cross-links are used, at least one of thedityrosine cross-links originates from a point mutation of an amino acidresidue to tyrosine. Furthermore, in some embodiments where DTcross-links are used, the methods described above further compriseintroducing one or more point mutations to tyrosine into the viralprotein or protein complex at one or more specific and/or identifiedregions before introducing dityrosine cross-links.

In some embodiments the methods of the invention further compriseperforming an assay to assess the ability of the engineered viralprotein or protein complex to bind to a neutralizing antibody, bind to abroadly neutralizing antibody, bind to and activate B cell receptors,elicit an antibody response in an animal, elicit a protective antibodyresponse in an animal, elicit production of neutralizing antibodies inan animal, elicit production of broadly neutralizing antibodies in ananimal, elicit a protective immune response in an animal, and/or elicitproduction of antibodies that recognize quaternary neutralizing epitopesin an animal.

In some embodiments the engineered viral proteins or protein complexesmade using the methods of the invention are useful as a vaccineimmunogens in animal subjects. In some embodiments the engineered viralproteins or protein complexes made using the methods of the inventionare useful as a vaccine immunogens in mammalian subjects. In someembodiments the engineered viral proteins or protein complexes areuseful as a vaccine immunogens in human subjects.

The methods of the present invention can be used to engineer proteinsfrom numerous different viruses. In some embodiments the viral proteinsor protein complexes are derived from a virus from the group consistingof Herpesvirales, Ligamenvirales, Mononegavirales, Nidovirales,Picornavirales, Lentiviruses, Human Immunodeficiency Viruses,Retroviruses, Orthomyxoviruses, Paramyxovirus, Influenza viruses,Poxviruses, Flaviviruses, Togaviruses, Coronaviruses, Rhabdoviruses,Bunyaviruses, Filoviruses, Reoviruses, Mononegavirales, Hepadnaviruses,and Hepatitis viruses. In some embodiments any viral protein may beengineered using the methods of the invention. In some embodiments theviral protein or protein complex to be engineered is a viral envelopeprotein or protein complex.

In some embodiments the engineered viral proteins or protein complexesof the invention are soluble. In some embodiments the engineered viralproteins or protein complexes of the invention form aggregates to alesser degree or not at all, for example during the production processor when stored at a high concentration, by comparison to a protein orprotein complex not so engineered.

In some embodiments the present invention provides pharmaceuticalcompositions comprising the engineered viral proteins or proteincomplexes of the invention. In some embodiments such compositionscomprise a pharmaceutically effective amount of the engineered viralproteins or protein complexes. In some embodiments such compositionsalso comprise a pharmaceutically acceptable carrier. In some embodimentssuch compositions also comprise an adjuvant.

In some embodiments, the present invention provides methods forstabilizing envelope proteins and protein complexes of pathogenicviruses to enhance their effectiveness as vaccine immunogens. In oneembodiment, the present invention provides methods by which tertiarystructures of proteins and/or quarternary structures of proteincomplexes (i.e. protein-protein interactions in a complex of two or moreproteins) can be stabilized by crosslinking, whereby the crosslinks arestable under physiologically relevant conditions, do not lead toaggregate formation of the proteins or protein complexes duringexpression or when they are stored in high concentrations, andstabilizes the folds of the proteins or protein complexes in particularconformations that can increase the effectiveness of the proteins orprotein complexes as vaccine immunogens—for example by stabilizingepitopes in such conformations that can be recognized by antibodiesand/or activate B cell receptors upon binding. In some such embodimentsthe crosslinks can be specifically directed to particular residueswithin the proteins or protein complexes, such as, for example, bydityrosine bonds, or the crosslinks can be directed to amino andsulfhydryl containing amino acid side chains. In some such embodimentsthe crosslinks can be zero-length, or may insert additional atoms andelements into the structure of the protein. In some embodiments, thepresent invention provides methods by which proteins or proteincomplexes can be oligomerized by oligomerization motifs that canstabilize protein complexes, and also stabilize the folds of theproteins in such protein complexes in particular conformations, such asthose that increase the effectiveness of the proteins or proteincomplexes as vaccine immunogens, for example by stabilizing epitopes inconformations that can be recognized by antibodies and activate B cellreceptors upon binding.

These and other embodiments of the invention are described throughoutthe present application, including in the Summary of Invention, DetailedDescription, Examples, and Claims sections of the application.Furthermore, the various embodiments described herein can be combinedand modified in various ways, as will be apparent to those of ordinaryskill in the art, and such combinations and modifications are within thescope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Analysis of dityrosine cross-linked HIV Env gp140 trimers. FIG.1A—Bar graph showing the results of a dityrosine (DT) specificspectrofluorometry experiment which was used to identify and quantify DTcrosslinks in wild type control (“WT control”) HIV Env gp140 protein andan engineered HIV Env gp140 protein having tyrosine substitution(s) inthe V1/V2 region (“mutant”), both before and after (+DT) dityrosinecrosslinking FIG. 1B—Left panel—Coomassie staining of the mutant HIV Envgp140 protein without (“−”) or with (“+”) DT cross-linking FIG. 1B—Rightpanel—Western blot of purified HIV Env gp140 without (“−”) or with (“+”)DT cross-linking Arrows indicate the locations of the monomeric andtrimeric forms.

FIG. 2. Binding of wild type HIV Env protomer and conformationallylocked HIV Env trimer to varying concentrations of the broadlyneutralizing antibody PG16 was measured by enzyme-linked immunosorbentassay (ELISA). The lower line on the graph represents binding of wildtype (WT) HIV Env protomer to varying concentrations of PG16, while theupper line represents binding of a conformationally locked HIV Envtrimer to varying concentrations of PG16.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides, in part, methods of engineering viralproteins and protein complexes, viral proteins and protein complexes soengineered, and pharmaceutical compositions comprising such engineeredviral proteins and protein complexes. Such engineered proteins may beuseful as conformationally-specific immunogens.

In one embodiment the present invention provides a method for producinga conformationally-specific vaccine immunogen, the method comprising:(a) obtaining a viral protein or protein complex in one or moreconformations that favor the elicitation of protective immune responses,(b) identifying one or more regions in the tertiary and/or quaternarystructure of the viral protein or protein complex in which theintroduction of one or more cross-links could stabilize the conformationof an antibody epitope (and/or could stabilize a conformation thatfavors the elicitation of a protective immune response), and (c)introducing into the viral protein or protein complex one or moretargeted cross-links at one or more of the regions identified in step(b) to form an engineered viral protein or protein complex, wherein theengineered viral protein or protein complex has one or more of thefollowing properties: (i) enhanced ability bind to a neutralizingantibody as compared to the viral protein or protein complex (i.e. ascompared to the viral protein or protein complex without or beforeintroduction of the cross-links), (ii) enhanced ability bind to abroadly neutralizing antibody as compared to the viral protein orprotein complex, (iii) enhanced ability bind to and activate B cellreceptors as compared to the viral protein or protein complex, (iv)enhanced ability to elicit an antibody response in an animal as comparedto the viral protein or protein complex, (v) enhanced ability to elicita protective antibody response in an animal as compared to the viralprotein or protein complex, (vi) enhanced ability to elicit productionof neutralizing antibodies in an animal as compared to the viral proteinor protein complex, (vii) enhanced ability to elicit production ofbroadly neutralizing antibodies in an animal as compared to the viralprotein or protein complex, (viii) enhanced ability to elicit aprotective immune response in an animal as compared to the viral proteinor protein complex, and (ix) enhanced ability to bind to and elicitproduction of antibodies that recognize quaternary neutralizing epitopesin an animal as compared to the viral protein or protein complex. Insome such embodiments the targeted cross-links are dityrosine (DT)cross-links.

In another embodiment the present invention provides a method forproducing a conformationally specific vaccine immunogen, the methodcomprising: (a) obtaining a viral protein or protein complex in one ormore conformations that favor the elicitation of protective immuneresponses, and (b) introducing into the viral protein or protein complexone or more cross-links that are stable under physiological conditions,wherein the engineered viral protein or protein complex has one or moreof the following properties: (i) enhanced ability bind to a neutralizingantibody as compared to the viral protein or protein complex (i.e. ascompared to the viral protein or protein complex without or beforeintroduction of the cross-links), (ii) enhanced ability bind to abroadly neutralizing antibody as compared to the viral protein orprotein complex, (iii) enhanced ability bind to and activate B cellreceptors as compared to the viral protein or protein complex, (iv)enhanced ability to elicit an antibody response in an animal as comparedto the viral protein or protein complex, (v) enhanced ability to elicita protective antibody response in an animal as compared to the viralprotein or protein complex, (vi) enhanced ability to elicit productionof neutralizing antibodies in an animal as compared to the viral proteinor protein complex, (vii) enhanced ability to elicit production ofbroadly neutralizing antibodies in an animal as compared to the viralprotein or protein complex, (viii) enhanced ability to elicit aprotective immune response in an animal as compared to the viral proteinor protein complex, and (ix) enhanced ability to bind to and elicitproduction of antibodies that recognize quaternary neutralizing epitopesin an animal as compared to the viral protein or protein complex. Insome such embodiments the cross-links are targeted to identified and/orselected positions within the protein or protein complex's tertiary orquaternary structure. In some such embodiments the targeted cross-linkscomprise dityrosine (DT) cross links.

In some embodiments where DT cross-links are used, at least one of thedityrosine cross-link originates from a point mutation of an amino acidresidue to tyrosine. Furthermore, in some embodiments where DTcross-links are used, the methods described above further compriseintroducing one or more point mutations to tyrosine into the viralprotein or protein complex at one or more specific and/or identifiedregions before introducing dityrosine cross-links.

In some embodiments the methods of the invention further compriseperforming an assay to assess the ability of the engineered viralprotein or protein complex to bind to a neutralizing antibody, bind to abroadly neutralizing antibody, bind to and activate B cell receptors,elicit an antibody response in an animal, elicit a protective antibodyresponse in an animal, elicit production of neutralizing antibodies inan animal, elicit production of broadly neutralizing antibodies in ananimal, elicit a protective immune response in an animal, and/or elicitproduction of antibodies that recognize quaternary neutralizing epitopesin an animal.

In some embodiments the engineered viral proteins or protein complexesmade using the methods of the invention are useful as a vaccineimmunogens in animal subjects. In some embodiments the engineered viralproteins or protein complexes made using the methods of the inventionare useful as a vaccine immunogens in mammalian subjects. In someembodiments the engineered viral proteins or protein complexes areuseful as a vaccine immunogens in human subjects.

The methods of the present invention can be used to engineer proteins orprotein complexes from numerous different viruses. In some embodimentsthe viral proteins or protein complexes are derived from a virus fromthe group consisting of Herpesvirales, Ligamenvirales, Mononegavirales,Nidovirales, Picornavirales, Lentiviruses, Human ImmunodeficiencyViruses, Retroviruses, Orthomyxoviruses, Paramyxovirus, Influenzaviruses, Poxviruses, Flaviviruses, Togaviruses, Coronaviruses,Rhabdoviruses, Bunyaviruses, Filoviruses, Reoviruses, Mononegavirales,Hepadnaviruses, and Hepatitis viruses. In some embodiments any viralprotein may be engineered using the methods of the invention. In someembodiments the viral protein or protein complex to be engineered is aviral envelope protein or protein complex.

In some embodiments the viral proteins or protein complexes and/or theengineered viral proteins or protein complexes of the invention aresoluble. In some embodiments the engineered viral proteins or proteincomplexes of the invention do not form aggregates, for example duringthe production process or when stored at a high concentration.

In some embodiments the present invention provides pharmaceuticalcompositions comprising the engineered viral proteins or proteincomplexes of the invention. In some embodiments such compositionscomprise a pharmaceutically effective amount of the engineered viralproteins or protein complexes. In some embodiments such compositionsalso comprise a pharmaceutically acceptable carrier. In some embodimentssuch compositions also comprise an adjuvant.

In some embodiments, the present invention provides methods forstabilizing envelope proteins and protein complexes of pathogenicviruses to enhance their effectiveness as vaccine immunogens. In oneembodiment, the present invention provides methods by which tertiarystructures of proteins and/or quarternary structures of proteincomplexes (i.e. protein-protein interactions in a complex of two or moreproteins) can be stabilized by crosslinking, whereby the crosslinks arestable under physiologically relevant conditions, do not lead toaggregate formation of the proteins or protein complexes duringexpression or when they are stored in high concentrations, andstabilizes the folds of the proteins or protein complexes in particularconformations that can increase the effectiveness of the proteins orprotein complexes as immunogens—for example by stabilizing epitopes insuch conformations that can be recognized by antibodies and/or activateB cell receptors upon binding. In some such embodiments the crosslinkscan be specifically directed to particular residues within the proteinsor protein complexes, such as, for example, by dityrosine bonds, or thecrosslinks can be directed to amino and sulfhydryl containing amino acidside chains. In some such embodiments the crosslinks can be zero-length,or may insert additional atoms and elements into the structure of theprotein. In some embodiments, the present invention provides methods bywhich proteins or protein complexes can be oligomerized byoligomerization motifs that can stabilize protein complexes, and alsostabilize the folds of the proteins in such protein complexes inparticular conformations, such as those that increase the effectivenessof the proteins or protein complexes as vaccine immunogens, for exampleby stabilizing epitopes in conformations that can be recognized byantibodies and activate B cell receptors upon binding.

In some embodiments the present invention provides a method forproducing an engineered viral protein or protein complex useful as avaccine immunogen, the method comprising introducing into a viralprotein or protein complex one or more cross-links, thereby forming anengineered viral protein or protein complex. In some such embodimentsthe engineered viral protein or protein complex is useful as a vaccineimmunogen in a vertebrate animal, such as in a mammal, or morespecifically a human.

In some embodiments of the present invention, cross-links are introducedto stabilize the engineered viral proteins or protein complexes in aconformation that counteracts conformational masking by the virus. Insome embodiments the crosslinks stabilize the engineered viral proteinsor protein complexes in a conformation that can bind to and activate a Bcell receptor. In some embodiments the crosslinks stabilize theengineered viral proteins or protein complexes in a conformation that iscapable of eliciting an antibody response in an animal. In someembodiments the crosslinks stabilize the engineered viral proteins orprotein complexes in a conformation that is capable of eliciting aneutralizing antibody response. In some embodiments the crosslinksstabilize the engineered viral proteins or protein complexes in aconformation that is capable of eliciting a broadly neutralizingantibody response. In some embodiments the crosslinks stabilize theengineered viral proteins or protein complexes in a conformation that iscapable of eliciting conformationally specific antibodies. In someembodiments the crosslinks stabilize the engineered viral proteins orprotein complexes in a conformation that is capable of elicitingantibodies that recognize quaternary epitopes. In some embodiments thecrosslinks stabilize the engineered viral proteins or protein complexesin a conformation that is capable of eliciting antibodies that recognizequaternary neutralizing epitopes. In some embodiments the crosslinksstabilize the engineered viral proteins or protein complexes in aconformation that is capable of eliciting antibodies that recognizemetastable epitopes. In some embodiments the crosslinks stabilize theengineered viral proteins or protein complexes in a conformation that iscapable of eliciting a broadly protective antibody response against avirus. In some embodiments the crosslinks stabilize the engineered viralproteins or protein complexes in a conformation that is capable ofeliciting a neutralizing immune response against a virus. In someembodiments the crosslinks stabilize the engineered viral proteins orprotein complexes in a conformation that is capable of eliciting abroadly neutralizing immune response against a virus. In someembodiments the crosslinks stabilize the engineered viral proteins orprotein complexes in a conformation that is capable of eliciting anenhanced humoral immune response in a mammal. In some embodiments thecrosslinks stabilize the engineered viral proteins or protein complexesin a conformation that is capable of eliciting a humoral immune that canprotect an individual from infection by a virus. In some embodiments thecrosslinks stabilize the engineered viral proteins or protein complexesin a conformation that can be bound by an antibody. In some embodimentsthe crosslinks stabilize the engineered viral proteins or proteincomplexes in a conformation that can be bound by a neutralizingantibody. In some embodiments the crosslinks stabilize the engineeredviral proteins or protein complexes in a conformation that can be boundby a broadly neutralizing antibody. In some embodiments the crosslinksstabilize the engineered viral proteins or protein complexes in aconformation that is thermostable. In some embodiments the crosslinksstabilize the engineered viral proteins or protein complexes in aconformation that has a prolonged shelf-life. In some embodiments thecrosslinks stabilize the engineered viral proteins or protein complexesin a conformation that has a prolonged life or half-life inside the bodyof a subject. In some embodiments the crosslinks stabilize theengineered viral proteins or protein complexes in such a way that theconformation isomer (i.e. the form of the protein having thecorrect/desired conformation) has a prolonged life or half-life insidethe body of a subject.

In some embodiments the engineered proteins or protein complexes of theinvention can bind to and activate a B cell receptor.

In some embodiments the engineered proteins or protein complexes of theinvention can elicit an antibody response in an animal, such as aneutralizing antibody response or a broadly neutralizing antibodyresponse. In some such embodiments the antibody response comprisesgeneration of conformationally-specific antibodies. In some suchembodiments the antibody response comprises generation of antibodiesthat recognize quaternary epitopes, such as quaternary neutralizingepitopes or QNEs. In some such embodiments the antibody responsecomprises generation of antibodies that recognize metastable epitopes.

In some embodiments the engineered proteins or protein complexes of theinvention can elicit a broadly protective antibody response against avirus. In some embodiments the engineered proteins or protein complexesof the invention can elicit a neutralizing immune response against avirus, such as a broadly neutralizing immune response against a virus.In some embodiments the engineered proteins or protein complexes of theinvention can elicit an enhanced humoral immune response in a mammal. Insome embodiments the engineered proteins or protein complexes of theinvention can elicit a humoral immune response that can protect ananimal subject (such as a mammalian subject, or a human subject) frominfection by a virus.

In some embodiments the engineered proteins or protein complexes of theinvention can bind to an antibody, such as a neutralizing antibody, or abroadly neutralizing antibody. In some embodiments the engineeredproteins or protein complexes of the invention preferentially bind toneutralizing antibodies or broadly neutralizing antibodies.

In some embodiments the engineered proteins or protein complexes of theinvention can bind to at least one neutralizing antibody and at leastone non-neutralizing antibody, and bind to the neutralizingantibody(ies) with an affinity that is higher than that with which theybind to the non-neutralizing antibody(ies).

In some embodiments of the invention described herein, the antibodiesthat bind to the engineered proteins or protein complexes of theinvention are monoclonal antibodies.

In some embodiments the crosslinks introduced into the engineered viralproteins or protein complexes of the invention stabilize folds in thestructure of the engineered viral protein or protein complex.

In some embodiments of the present invention crosslinks are introducedinto the viral proteins or protein complexes after the viral proteins orprotein complexes are fully folded. In particular, in some embodimentsof the present invention crosslinks are introduced into the viralproteins or protein complexes after the viral proteins or proteincomplexes are fully folded into a conformation that favors: (i) theelicitation of a protective immune response, or (ii) binding of aneutralizing antibody, or (iii) binding of a broadly neutralizingantibody, or (iv) binding and activation of B cell receptors, or (v) theelicitation of an antibody response in an animal, or (vi) elicitation ofa protective antibody response in an animal, or (vii) elicitation ofneutralizing antibodies in an animal, or (vii) elicitation of broadlyneutralizing antibodies in an animal, or (viii) elicitation of aprotective immune response in an animal, or (ix) elicitation ofantibodies that recognize quaternary neutralizing epitopes in an animal,so as to “lock” the protein or protein complex into such a conformation.

In some embodiments the crosslinks stabilize the tertiary structure ofan engineered viral protein or protein complex. In some embodiments thecrosslinks stabilize the quaternary structure of an engineered viralprotein complex. In some embodiments the crosslinks stabilize both thetertiary and quaternary structure of an engineered viral proteincomplex.

In some embodiments the engineered viral proteins or protein complexesof the invention do not form aggregates in solution. In some embodimentsthe engineered viral proteins or protein complexes of the invention donot form aggregates when stored in solution at high concentration.

In some embodiments the engineered viral proteins or protein complexesof the invention have cross-links that are thermostable.

In some embodiments the engineered viral proteins or protein complexesof the invention have cross-links are not toxic.

In some embodiments the engineered viral proteins or protein complexesof the invention have cross-links that are targeted cross-links, ornon-targeted cross-links, or reversible cross-links, or irreversiblecross-links, or crosslinks formed by use of homo-bifunctionalcrosslinking agents, or crosslinks formed by use of hetero-bifunctionalcrosslinking agents, or crosslinks formed by use of reagents that reactwith amine groups, or crosslinks formed by use of reagents that reactwith thiol groups, or crosslinks formed by use of reagents that arephotoreactive, or crosslinks formed between amino acid residues, orcrosslinks formed between mutated amino acid residues incorporated intothe structure of the proteins or protein complexes, or oxidativecrosslinks, or dityrosine bonds, or glutaraldehdye cross-links, or anycombination thereof. In some embodiments the engineered viral proteinsor protein complexes of the invention do not have glutaraldehydecross-links. In some embodiments the engineered viral proteins orprotein complexes of the invention do not have any disulfide bonds. Insome embodiments the engineered viral proteins or protein complexes ofthe invention do not have any artificially introduced disulfide bonds.

In some embodiments the engineered viral proteins or protein complexesare derived from a virus from the group consisting of Herpesvirales,Ligamenvirales, Mononegavirales, Nidovirales, Picornavirales,Lentiviruses, Human Immunodeficiency Viruses, Retroviruses,Orthomyxoviruses, Paramyxovirus, Influenza viruses, Poxviruses,Flaviviruses, Togaviruses, Coronaviruses, Rhabdoviruses, Bunyaviruses,Filoviruses, Reoviruses, Mononegavirales, Hepadnaviruses, and Hepatitisviruses. In some embodiments, the engineered viral proteins or proteincomplexes are derived from viral envelope proteins or protein complexes.In some such embodiments the viral envelope protein or protein complexis a Type I, Type II, or Type III Fusion protein.

In some embodiments of the invention the viral proteins or proteincomplexes, and/or the engineered viral proteins or protein complexes,are isolated. In some embodiments of the invention the viral proteins orprotein complexes, and/or the engineered viral proteins or proteincomplexes, are purified. In some embodiments of the invention the viralproteins or protein complexes, and/or the engineered viral proteins orprotein complexes, are isolated. In some embodiments of the inventionthe viral proteins or protein complexes, and/or the engineered viralproteins or protein complexes, are soluble. In some embodiments of theinvention the viral proteins or protein complexes, and/or the engineeredviral proteins or protein complexes, are proteolytically cleaved.

In some embodiments the present invention provides methods of producinga conformationally-specific immunogen, or methods or producing anengineered viral protein or protein complex, wherein the methodscomprise incorporating an engineered viral protein or protein complexinto a composition, such as a pharmaceutical composition. In some suchmethods the pharmaceutical composition comprises a pharmaceuticallyeffective amount of the engineered viral protein or protein complex. Insome such methods the pharmaceutical composition comprises apharmaceutically acceptable carrier. In some such methods thepharmaceutical composition comprises an adjuvant. In some such methodsthe pharmaceutical composition comprises a pharmaceutically effectiveamount of the engineered viral protein or protein complex and apharmaceutically acceptable carrier. In some such methods thepharmaceutical composition comprises a pharmaceutically effective amountof the engineered viral protein or protein complex and apharmaceutically acceptable carrier and an adjuvant.

In some embodiments the present invention provides compositionscomprising an engineered viral protein or protein complex as describedherein. In some embodiments the present invention providespharmaceutical compositions comprising an engineered viral protein orprotein complex as described herein. In some embodiments the presentinvention provides pharmaceutical compositions comprising apharmaceutically effective amount of an engineered viral protein orprotein complex as described herein. In some embodiments the presentinvention provides pharmaceutical compositions comprising a anengineered viral protein or protein complex as described herein and apharmaceutically acceptable carrier. In some embodiments the presentinvention provides pharmaceutical compositions comprising a anengineered viral protein or protein complex as described herein and anadjuvant. In some embodiments the present invention providespharmaceutical compositions comprising a pharmaceutically effectiveamount of an engineered viral protein or protein complex as describedherein and a pharmaceutically acceptable carrier. In some embodimentsthe present invention provides pharmaceutical compositions comprising apharmaceutically effective amount of an engineered viral protein orprotein complex as described herein, a pharmaceutically acceptablecarrier, and an adjuvant.

In some embodiments the present invention provides engineered viralproteins or protein complexes, or compositions comprising engineeredviral proteins or protein complexes, wherein the engineered viralproteins or protein complexes have one or more of properties selectedfrom the group consisting of: (i) enhanced ability bind to aneutralizing antibody, (ii) enhanced ability bind to a broadlyneutralizing antibody, (iii) enhanced ability bind to and activate Bcell receptors, (iv) enhanced ability to elicit an antibody response inan animal, (v) enhanced ability to elicit a protective antibody responsein an animal, (vi) enhanced ability to elicit production of neutralizingantibodies in an animal, (vii) enhanced ability to elicit production ofbroadly neutralizing antibodies in an animal, (viii) enhanced ability toelicit a protective immune response in an animal, and (ix) enhancedability to elicit production of antibodies that recognize quaternaryneutralizing epitopes in an animal.

In some embodiments the present invention provides methods of producinga conformationally-specific vaccine immunogen, or methods of producingan engineered viral protein or protein complex, wherein the methodscomprise performing an assay to assess the ability of the engineeredviral protein or protein complex to bind to a neutralizing antibody,bind to a broadly neutralizing antibody, bind to and activate B cellreceptors, elicit an antibody response in an animal, elicit a protectiveantibody response in an animal, elicit production of neutralizingantibodies in an animal, elicit production of broadly neutralizingantibodies in an animal, elicit a protective immune response in ananimal, and/or elicit production of antibodies that recognize quaternaryneutralizing epitopes in an animal.

As used herein the terms “protein” and “polypeptide” are usedinterchangeably, unless otherwise stated. As used herein the term“protein complex” refers to an assembly of two or more proteins. Unlessotherwise stated, all description herein that relates to proteinsapplies equally to protein complexes, and vice versa. As used herein theterm “engineered” in relation to proteins and/or protein complexesgenerally refers to those that include cross-links, typically as theresult of the introduction of cross-links in order to stabilize theprotein or protein complex in a desirable conformation. Unless otherwisestated, all description herein that relates to proteins or proteincomplexes (including, but not limited to, that related to methods ofmaking and using such proteins), relates equally to engineeredproteins/protein complexes and non-engineered proteins/protein complexes(e.g. those with no cross-links added or before addition of cross-links)and to all homologs, orthologs, analogs, derivatives, mutant forms,fragments, chimeras, fusion proteins etc. thereof. The terms “protein”and “protein complex” include naturally occurring viral proteins andprotein complexes, and viral proteins and protein complexes that havebeen altered in some way, such as by recombinant means, chemical means,or any other means. Such proteins and protein complexes include, but arenot limited to, those from viruses that are pathogenic, such as thosethat are pathogenic to humans or other animals, and for which it isdesirable to engineer such proteins and protein complexes, for examplein order to enhance the immunogenicity of the protein or protein complexand/or to enhance the capability of the protein or protein complex toelicit a humoral immune responses in an animal, such as a human or othermammal.

In some embodiments the methods and compositions of the invention can beused with any suitable viral protein or protein complex. In someembodiments the viral proteins and protein complexes are viral envelopeproteins or protein complexes. Many viruses have a viral envelopecovering their protein capsid. The envelope of such viruses typicallycomprises host cell phospholipids and proteins, and further includesviral proteins and/or protein complexes—which typically compriseglycoproteins. The viral envelope mediates many of the processesinvolved in viral entry into host cells and infection. For example,surface viral envelope glycoproteins of a virus particle can bind tohost cell receptor molecules on the host cell's membrane and affectfusion of the viral envelope with the host cell's membrane allowingsubsequent entry of the capsid and viral genome and infection of thehost cell. Because viral envelope proteins are on the surface of viralparticles, and are therefore accessible, they are considered among thebest targets for vaccine immunogen design and development.

Viral proteins and/or protein complexes used in accordance with thepresent invention can have, or can be derived from, the nucleotideand/or amino acid sequences of any suitable virus proteins or proteincomplexes known in the art, and can have, or be derived from nucleotideand/or amino acid sequences that have at least about 85%, or about 90%,or about 95%, or about 98% sequence identity to any such knownsequences, or to any groups, subgroups, families, subfamilies, types,subtypes, genera, species, strains, and clades, etc. of any knownviruses. As used in the present specification the terms “about” and“approximately,” when used in relation to numerical values, meanwithin + or −20% of the stated value.

In some embodiments, the invention provides fragments of the proteins orprotein complexes of the invention, such as those comprising, consistingessentially of, or consisting of at least about 10 amino acids, 20 aminoacids, 50 amino acids, 100 amino acids, 200 amino acids, 500 aminoacids, 1000 amino acids, 2000 amino acids, or 5000 amino acids.

Derivatives or analogs of the proteins of the invention include thosemolecules comprising regions that are substantially homologous to aprotein or fragment thereof (e.g., in various embodiments, those havingat least about 40% or 50% or 60% or 70% or 80% or 90% or 95% identitywith an amino acid or nucleic acid sequence of the invention whenaligned using any suitable method known to one of ordinary skill in theart, such as, for example, using a computer homology program known inthe art) or whose encoding nucleic acid is capable of hybridizing to acoding nucleic acid sequence of a protein of the invention, under highstringency, moderate stringency, or low stringency conditions.

In some embodiments one or more amino acid residues within a protein orprotein complex can be substituted with another amino acid. For example,one or more amino acid residues can be substituted by another amino acidhaving a similar polarity and that may acts as a functional equivalent,resulting in a silent alteration. In some embodiments substitutions foran amino acid within the sequence may be selected from other members ofthe class to which the amino acid belongs e.g. to create a conservativesubstitution. For example, the nonpolar (hydrophobic) amino acidsinclude alanine, leucine, isoleucine, valine, proline, phenylalanine,tryptophane and methionine. The polar neutral amino acids includeglycine, serine, threonine, cysteine, tyrosine, asparagine, andglutamine. The positively charged (basic) amino acids include arginine,lysine and histidine. The negatively charged (acidic) amino acidsinclude aspartic acid and glutamic acid. Such substitutions aregenerally understood to be conservative substitutions.

Proteins and protein complexes can be produced by any methods known toone of ordinary skill in the art, and manipulations of such proteins orprotein complexes can occur or be made at the nucleic acid orprotein/amino acid level. For example, a cloned nucleotide sequenceencoding a protein or protein complex can be modified by any of numerousstrategies known to one of ordinary skill in the art.

Chimeric proteins can be made by any method known to one of ordinaryskill in the art, and may comprise, for example, one or several proteinsof the invention, such as those that have been engineered enhance theirimmunogenicit, and/or any fragment, derivative, or analog thereof(preferably consisting of at least a domain of a polypeptide, protein,or protein complex to be engineered, or at least 6, and preferably atleast 10 amino acids of the protein) joined at its amino- orcarboxy-terminus via a peptide bond to an amino acid sequence of adifferent protein. In some embodiments such chimeric proteins can beproduced by any method known to one of ordinary skill in the art,including, but not limited to, recombinant expression of a nucleic acidencoding a chimeric protein (e.g. comprising a first coding sequencejoined in-frame to a second coding sequence); ligating the appropriatenucleic acid sequences encoding the desired amino acid sequences to eachother in the proper coding frame, and expressing the chimeric product.In another embodiment protein synthetic techniques can be used togenerate any protein (including chimeric protein), for example by use ofa peptide synthesizer.

In some embodiments viral proteins and/or protein complexes can beengineered in such a way that they are capable of eliciting a humoralimmune responses that may protect, or help protect, an individual frominfection by a particular virus. The phrase “capable of eliciting ahumoral immune response,” as used herein, can refer, in someembodiments, to a protein or protein complex that can cause cells of theimmune system to produce antibodies that bind to the proteins orcomplexes. In some embodiments the antibodies bind with dissociationconstants (KD) of less than 5 times 10.sup.-2 M, 10.sup.-2 M, 5 times10.sup.-3 M, 10.sup.-3 M, 5 times 10.sup.-4 M, 10.sup.-4 M, 5 times10.sup.-5 M, 10.sup.-5 M, 5 times 10.sup.-6 M, 10.sup.-6 M, 5 times10.sup.-7 M, 10.sup.-7 M, 5 times 10.sup.-8 M or 10.sup.-8 M, 5 times10.sup.-9 M, 10.sup.-9 M, 5 times 10.sup.-10 M, 10.sup.-10 M, 5 times10.sup.-11 M, 10.sup.-11 M, 5 times 10.sup.-12 M, 10.sup.-12 M, 5 times10.sup.-13 M, 10.sup.-13 M, 5 times 10.sup.-14 M, 10.sup.-14 M, 5 times10.sup.-15 M, or 10.sup.-15 M, with an off rate (k.sub.off) of less thanor equal to 5 times 10.sup.-2 sec.sup.-1, 10.sup.-2 sec.sup.-1, 5 times10.sup.-3 sec.sup.-1, 10.sup.-3 sec.sup.-1, 5 times 10.sup.-4sec.sup.-1, 10.sup.-4 sec.sup.-1, 5 times 10.sup.-5 sec.sup.-1, or10.sup.-5 sec.sup.-1, 5 times 10.sup.-6 sec.sup.-1, 10.sup.-6sec.sup.-1, 5 times 10.sup.-7 sec.sup.-1, or 10.sup.-7 sec.sup.-1,and/or with an on rate (k.sub.on) of greater than or equal to 10.sup.3M.sup.-1 sec.sup.-1, 5 times 10.sup.3 M.sup.-1 sec.sup.-1, 10.sup.4M.sup.-1 sec.sup.-1, times 10.sup.4 M.sup.-1 sec.sup.-1, 10.sup.5M.sup.-1 sec.sup.-1, 5 times 10.sup.5 M.sup.-1 sec.sup.-1, 10.sup.6M.sup.-1 sec.sup.-1, or 5 times 10.sup.6 M.sup.-1 sec.sup.-1, or10.sup.7 M.sub.-1 sec.sub.-1.

Proteins and protein complexes may also be altered by adding or deletingamino acid residues, by adding or removing post-translationalmodifications, by adding or removing chemical modifications orappendixes, and/or by introducing any other mutations or modificationsknown to those of ordinary skill in the art.

Included within the scope of the invention are proteins and proteincomplexes that are modified during or after translation or synthesis,for example, by crosslinking, glycosylation, acetylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, or buy any other means known in the art.For example, in some embodiments the proteins and protein complexes maybe subjected to chemical cleavage by cyanogen bromide, trypsin,chymotrypsin, papain, V8 protease, NaBH4, acetylation, formylation,oxidation, reduction, metabolic synthesis in the presence oftunicamycin, etc.

The proteins and protein complexes of the invention can be made by anysuitable means known in the art, including recombinant means andchemical synthesis means. In addition, proteins and protein complexes ofthe invention can be engineered for enhanced immunogenicity using anysuitable means known in the art. For example, a peptide corresponding toa portion of a protein or protein complex can be synthesized by use of apeptide synthesizer. Furthermore, if desired, artificial, synthetic, ornon-classical amino acids or chemical amino acid analogs can be used tomake the proteins and protein complexes of the invention or introducedinto the proteins and protein complexes of the invention. Non-classicalamino acids include, but are not limited to, the D-isomers of the commonamino acids, fluoro-amino acids, and “designer” amino acids such asβ-methyl amino acids, Cγ-methyl amino acids, Nγ-methyl amino acids, andamino acid analogs in general. Additional non-limiting examples ofnon-classical amino acids include, but are not limited to:α-aminocaprylic acid, Acpa; (S)-2-aminoethyl-L-cysteine/HCl, Aecys;aminophenylacetate, Afa; 6-amino hexanoic acid, Ahx; γ-amino isobutyricacid and α-aminoisobytyric acid, Aiba; alloisoleucine, Aile;L-allylglycine, Alg; 2-amino butyric acid, 4-aminobutyric acid, andα-aminobutyric acid, Aba; p-aminophenylalanine, Aphe; b-alanine, Bal;p-bromophenylalaine, Brphe; cyclohexylalanine, Cha; citrulline, Cit;β-chloroalanine, Clala; cycloleucine, Cle; p-cholorphenylalanine, Clphe;cysteic acid, Cya; 2,4-diaminobutyric acid, Dab; 3-amino propionic acidand 2,3-diaminopropionic acid, Dap; 3,4-dehydroproline, Dhp;3,4-dihydroxylphenylalanine, Dhphe; p-fluorophenylalanine, Fphe;D-glucoseaminic acid, Gaa; homoarginine, Hag; δ-hydroxylysine/HCl, Hlys;DL-β-hydroxynorvaline, Hnvl; homoglutamine, Hog; homophenylalanine,Hoph; homoserine, Hos; hydroxyproline, Hpr; p-iodophenylalanine, Iphe;isoserine, Ise; α-methylleucine, Mle;DL-methionine-S-methylsulfoniumchloide, Msmet; 3-(1-naphthyl)alanine,1Nala; 3-(2-naphthyl)alanine, 2Nala; norleucine, Nle; N-methylalanine,Nmala; Norvaline, Nva; O-benzylserine, Obser; O-benzyltyrosine, Obtyr;O-ethyltyrosine, Oetyr; O-methylserine, Omser; O-methylthreonine, Omthr;O-methyltyrosine, Omtyr; Ornithine, Orn; phenylglycine; penicillamine,Pen; pyroglutamic acid, Pga; pipecolic acid, Pip; sarcosine, Sar;t-butylglycine; t-butylalanine; 3,3,3-trifluoroalanine, Tfa;6-hydroxydopa, Thphe; L-vinylglycine, Vig;(−)-(2R)-2-amino-3-(2-aminoethylsulfonyl)propanoic aciddihydroxochloride, Aaspa; (2S)-2-amino-9-hydroxy-4,7-dioxanonanoic acid,Ahdna; (2S)-2-amino-6-hydroxy-4-oxahexanoic acid, Ahoha;(−)-(2R)-2-amino-3-(2-hydroxyethylsulfonyl)propanoic acid, Ahsopa;(−)-(2R)-2-amino-3-(2-hydroxyethylsulfanyl)propanoic acid, Ahspa;(2S)-2-amino-12-hydroxy-4,7,10-trioxadodecanoic acid, Ahtda;(2S)-2,9-diamino-4,7-dioxanonanoic acid, Dadna;(2S)-2,12-diamino-4,7,10-trioxadodecanoic acid, Datda;(S)-5,5-difluoronorleucine, Dfnl; (S)-4,4-difluoronorvaline, Dfnv;(3R)-1-1-dioxo-[1,4]thiaziane-3-carboxylic acid, Dtca;(S)-4,4,5,5,6,6,6-heptafluoronorleucine, Hfnl;(S)-5,5,6,6,6-pentafluoronorleucine, Pfnl;(S)-4,4,5,5,5-pentafluoronorvaline, Pfnv; and(3R)-1,4-thiazinane-3-carboxylic acid, Tca. Furthermore, the amino acidcan be D (dextrorotary) or L (levorotary). For a review of classical andnon-classical amino acids, see Sandberg et al., 1998 (Sandberg et al.,1998. New chemical descriptors relevant for the design of biologicallyactive peptides. A multivariate characterization of 87 amino acids. JMed Chem 41(14): pp. 2481-91).

Any suitable method known in the art may be used to generate or obtainproteins and protein complexes according to the present invention.Similarly, the proteins and protein complexes of the invention may beisolated or purified using any suitable method known in the art. Suchmethods include, but are not limited to, chromatography (e.g. ionexchange, affinity, and/or sizing column chromatography), ammoniumsulfate precipitation, centrifugation, differential solubility, or byany other technique for the purification of proteins known to one ofordinary skill in the art. The proteins and protein complexes may bepurified from any source that produces such proteins/complexes. Forexample, proteins and protein complexes may be purified from sourcesincluding, prokaryotic, eukaryotic, mono-cellular, multi-cellular,animal, plant, fungus, vertebrate, mammalian, human, porcine, bovine,feline, equine, canine, avian, tissue culture cells, and any othersource. The degree of purity may vary, but in various embodiments, thepurified protein is provided in a form in which is it comprises morethan about 10%, 20%, 50%, 75%, 85%, 95%, 99%, or 99.9% of the totalprotein.

In some embodiments point mutations can be introduced into proteinsand/or protein complexes to stabilize particular conformations. In someembodiments proteins may be deglycosylated, dephosphorylated, orotherwise chemically or enzymatically treated/altered to render themmore immunogenic, and capable of generating neutralizing and broadlyneutralizing immune responses against viral epitopes.

In embodiments where mutations are introduced into a protein or proteincomplex, the protein(s) can be micro-sequenced to determine a partialamino acid sequence. In some embodiments the partial amino acid sequencecan then be used together with, for example, library screening andrecombinant nucleic acid methods known in the art, for example toisolate clones having, or for introduction of, desired mutations.

In some embodiments the proteins and protein complexes of the inventionmay be isolated and purified from other proteins, or any otherundesirable products, by standard methods including, but not limited to,chromatography (e.g., sizing column chromatography, glycerol gradients,affinity), centrifugation, or by any other standard technique for thepurification of proteins. In specific embodiments it may be necessary toseparate proteins that are not part of one or more stabilized proteinsor protein complexes of the invention (e.g. that were not cross-linked),but that may, for example, homo- or heterodimerize with other proteins.Such separation may be achieved by any means known in the art,including, but not limited to, separation methods that use detergentsand/or reducing agents.

The yield of engineered proteins and protein complexes of the inventioncan be determined by any means known in the art, for example, bycomparing the amount of engineered proteins and/or protein complexesproduced as compared to the amount of the starting material (i.e. thenon-engineered proteins or protein complexes). Protein concentrationsare determined by standard procedures, such as, for example, Bradford orLowrie protein assays. The Bradford assay is compatible with reducingagents and denaturing agents (Bradford, M, 1976. Anal. Biochem. 72:248). The Lowry assay has better compatibility with detergents and thereaction is more linear with respect to protein concentrations andread-out (Lowry, O J, 1951. Biol. Chem. 193: 265).

In some embodiments proteins and/or protein complexes are obtainedand/or isolated in a conformation that favors the elicitation of aprotective immune response, and are subsequently cross-linked in orderto stabilize such conformation. Proteins and/or protein complexes may beobtained and/or isolated in conformations that favor the elicitation ofa protective immune response by any suitable method known in the art,including, for example, but not limited to, standard proteinpurification methods, such as ion exchange and size exclusionchromatography, and affinity chromatography. As further non-limitingexamples, proteins and protein complexes to be isolated may be expressedin the presence of, or co-expressed with, binding compounds, peptides,or proteins that stabilize the conformation of the proteins and proteincomplexes to be isolated when so bound. As further non-limitingexamples, proteins and protein complexes to be isolated may be expressedin high or low ionic media, or isolated in high or low ionic buffers orsolutions by the methods described herein. Proteins and proteincomplexes to be isolated may also be isolated at one or moretemperatures that favor preservation of the desired conformation.Proteins and protein complexes may also be isolated over a period oftime that diminishes the degree to which a preparation would have lostthe desired conformation. The degree to which a preparation of proteinsor protein complexes retains one or more conformations that favor theelicitation of protective immune responses may be assayed by anysuitable method known in the art, including, for example, but notlimited to, biochemical, biophysical, immunologic, and virologicanalyses. Such assays include, for example, but are not limited to,immunoprecipation, ELISA, or Enzyme-linked immunosorbent spot (ELISPOT)assays to analyze, for example, binding to protective or neutralizing orbroadly neutralizing antibodies or binding proteins; binding tonon-protective, non-neutralizing, or weakly protective or neutralizingantibodies or binding proteins; crystallographic analysis, includingco-crystallization with antibodies, sedimentation, analyticalultracentrifugation, dynamic light scattering (DLS), electron microscopy(EM), cryo-EM tomography, calorimetry, surface plasmon resonance (SPR),fluorescence resonance energy transfer (FRET), circular dichroismanalysis, and small angle x-ray scattering; neutralization assays ofimmune sera following immunization with proteins or protein complexes;antibody-dependent cellular cytotoxicity assays of immune sera followingimmunization with proteins or protein complexes; and virologic challengestudies in animals, and passive transfer assays.

Proteins and/or protein complexes of the invention may be stabilized byintra- and/or intermolecular crosslinking. Intramolecular crosslinkingmay stabilize the folds of particular protein conformations, andintermolecular crosslinking may stabilize both protein-proteininteractions and the folds of particular protein conformations, such asthose in which the proteins and protein complexes of the presentinvention have the desired immunogenic properties.

Crosslinks may include, but are not limited to, reversible crosslinksresulting from the use of homo- and hetero-bifunctional crosslinkingagents that react with amine and/or thiol groups, photoreactivecrosslink reagents, any crosslinks that may form between non-classicalamino acids incorporated into the structure of a protein or proteincomplex, and any oxidative crosslinks, such as, but not limited to,dityrosine cross-links/bonds. Irreversible crosslinks, as used in thecontext of this application, include those that are not dissolved underphysiologically relevant conditions, and do not lead to aggregateformation during expression or when stored in high concentrations.Disulfide bonds are not irreversible cross-links. Rather they arereversible cross-links and may dissolve under physiologically relevantconditions and/or lead to aggregate formation during protein expressionand/or production or when stored in high concentrations.

The crosslinks may be targeted to specific sites in the structure ofproteins and/or protein complexes in order to achieve the desiredimmunogenic properties. Alternatively, proteins an protein complexeswith the desired crosslinks may be isolated from a mixture ofcrosslinked and uncrosslinked proteins with and without the desiredmodifications, for example based on chemical, physical, and/orfunctional characteristics. Such characteristics may include, forexample, molecular weight, molecular volume, any and all chromatographicproperties, mobility in any all forms of electrophoresis, and any andall antigenic and biochemical characteristics, fluorescence and any andall other biophysical characteristics, solubility in aqueous solutions,(organic) solvents, and/or hybrid solutions in the presence or absenceof other molecules in solution (e.g. ions) at different concentrations,affinity to mono- and/or polyclonal antibodies, affinity to receptors,other proteins, DNA, RNA, lipids, other bio- and non-bio-organicmolecules and complexes, inorganic molecules and complexes, ions, anyand all structural characteristics, enzymatic, immunological, tissueculture, diagnostic, pharmaceutical, and any other activity oractivities, and any other characteristics that are known to one ofordinary skill in the art.

A wide variety of methods of crosslinking proteins intra- andinter-molecularly are known in the art, including those havingcross-links with varying lengths of spacer arms, and those with andwithout fluorescent and functional groups for purification. Such methodsinclude, but are not limited to, the use of heterobifunctionalcrosslinkers (e.g. succinimidyl acetylthioacetate (SATA),trans-4-(maleimidylmethyl)cyclohexane-1-carboxylate (SMCC), andsuccinimidyl 3-(2-pyridyldithio)propionate (SPDP)), homobifunctionalcrosslinkers (e.g. succinimidyl 3-(2-pyridyldithio)propionate),photoreactive crosslinkers (e.g. 4-azido-2,3,5,6-tetrafluorobenzoicacid, STP ester, sodium salt (ATFB, STP ester),4-azido-2,3,5,6-tetrafluorobenzoic acid, succinimidyl ester (ATFB, SE),4-azido-2,3,5,6-tetrafluorobenzyl amine, hydrochloride,benzophenone-4-isothiocyanate, benzophenone-4-maleimide,4-benzoylbenzoic acid, succinimidyl ester,N-((2-pyridyldithio)ethyl)-4-azidosalicylamide (PEAS; AET), thiolreactive crosslinkers (e.g. maleimides and iodoacetamides), aminereactive crosslinkers (e.g. glutaraldyde, bis(imido esters),bis(succinimidyl esters), diisocyanates and diacid chlorides). Becausethiol groups are highly reactive and relatively rare in most proteins bycomparison to amine groups, thiol-reactive crosslinking may be used insome embodiments. In cases where thiol groups are missing or not presentat appropriate sites in the structures of proteins and proteincomplexes, they can be introduced using one of several thiolationmethods. For examples, Succinimidyltrans-4-(maleimidylmethyl)cyclohexane-1-carboxylate can be used tointroduce thiol-reactive groups at amine sites.

Several oxidative crosslinks are known, such as disulfide bonds (whichform spontaneously and are pH and redox sensitive), and dityrosine bonds(which are highly stable, and irreversible under physiologicalconditions).

Therapeutic proteins are generally complex, heterogeneous, and subjectto a variety of enzymatic or chemical modifications during expression,purification, and long-term storage. Because they are often lyophilizedor stored and administered to patients at relatively highconcentrations, aggregate formation is often a problem, as it reducesmanufacturing yields and denatures the structure of the complex so thathumoral immune responses to conformationally masked epitopes are lesslikely to yield broadly neutralizing antibodies.

Engineering cystine side-chains by point mutation to positions wheredisulfide bonds will form stabilizes the HIV gp120 and gp41 proteins,and fragments thereof have been stabilized for the purpose of developingan immunogen capable of eliciting broadly neutralizing and/or broadlyneutralizing humoral immune responses (Beddows et al., 2006.Construction and Characterization of Soluble, Cleaved, and StabilizedTrimeric Env Proteins Based on HIV Type I Env Subtype A. AIDS Res HumRetroviruses 22(6): 569-579; Farzan et al., 1998. Stabilization of HumanImmunodeficiency Virus Type 1 Envelope Glycoprotein Trimers by DisulfideBonds Introduced into the gp41 Glycoprotein Ectodomain. J Virol 72(9):7620-25). Disulfide bonds are, however, known to be pH sensitive and tobe dissolved under certain redox conditions, and the preventative and/ortherapeutic utility of proteins and/or protein complexes engineered withdisulfide crosslinks, for example to be used as immunogens in vivo, maytherefore be compromised. Furthermore, undesired disulfide bonds oftenform between proteins with free sulfhydryl groups that mediate aggregateformation (see, for example, Harris R J et al. 2004, Commercialmanufacturing scale formulation and analytical characterization oftherapeutic recombinant antibodies. Drug Dev Res 61 (3): 137-154;Costantino & Pikal (Eds.), 2004. Lyophilization of Biopharmaceuticals,editors Costantino & Pekal. Lyophilization of Biopharmaceuticals.Series: Biotechnology: Pharmaceutical Aspects II, see pages 453-454;Tracy et al., 2002, U.S. Pat. No. 6,465,425), which has also beenreported as a problem with HIV gp120 and gp41 (Jeffs et al. 2004.Expression and characterisation of recombinant oligomeric envelopeglycoproteins derived from primary isolates of HIV-1.Vaccine22:1032-1046; Schulke et al., 2002. Oligomeric and conformationalproperties of a proteolytically mature, disulfide-stabilized humanimmunodeficiency virus type 1 gp140 envelope glycoprotein. J Virol76:7760-7776).

An alternative means of cross-linking proteins involves the formation ofdityrosine (DT) bonds. Dityrosine crosslinking introduces one or morecovalent carbon-carbon bonds into proteins or protein complexes. Thisprovides a method for stabilizing proteins, protein complexes, andconformations, by introduction of intra- and/or inter-polypeptidedi-tyrosine bonds while maintaining their structural and functionalintegrity (Marshall et al., U.S. Pat. Nos. 7,037,894 & 7,445,912).Creating covalent bonds at specific, targeted locations within proteinscan reinforce particular 3D-arrangements of the protein structure andcan provide a high degree of stabilization.

The minimally altering, and zero-length DT crosslink is not hydrolyzedunder physiological conditions, and has been demonstrated to maintainproteins' structural integrity by liquid chromatography/massspectrometry (LC/MS). Dityrosine crosslinks are known to be safe, asthey form naturally in vivo, both in the context of proteins evolved toutililze their specific characteristics (e.g. Elvin C M et al. 2005,Nature 437:999-1002; Tenovuo J & Paunio K 1979, Arch Oral Biol.;24(8):591-4), and as a consequence of non-specific protein oxidation(Giulivi et al. 2003, Amino Acids 25(3-4):227-32), and as they arepresent in large quantities in some of our most common foods: DT bondsform the structure of wheat gluten—the quarternary protein structurecomprising the glutenin subunits—e.g. in bread dough during mixing andbaking (Tilley et al. 2001, Agric. Food Chem 49, 2627).

Dityrosine bonds do not form spontaneously in vitro. Rather, theenzymatic crosslink reaction is carried out under optimized conditionsto preserve protein structure and function. Therefore, non-specificbonding/aggregation does not occur (as compared to free-sulfhydrylgroups), and therefore large-scale manufacturing of a DT stabilizedimmunogen may be economically more feasible.

Tyrosyl side-chains are present in many redox enzymes, and catalysis ofthe enzyme-specific reactions often involves tyrosyl radicals that arelong-lived and have comparatively low reactivity. Under optimizedconditions radical formation is specific to tyrosyl side-chains. Inclose proximity to each tyrosyl side chains undergo radical coupling andform a covalent, carbon-carbon bond. Tyrosyl radicals that do not reactrevert to non-radicalized tyrosyl side-chains (Malencik & Anderson,2003. Dityrosine as a product of oxidative stress and fluorescent probe.Amino Acids 25: 233-247). Therefore, tyrosyl side-chains must besituated in close proximity to form DT bonds, either within a singlefolded polypeptide chain, or on closely interacting protein domainswithin a complex. Because a Cα-Cα separation of approximately 5-8 Å isprerequisite to bond formation (Brown et al., 1998. Determiningprotein-protein interactions by oxidative cross-linking of aglycine-glycine-histidine fusion protein. Biochemistry 37, 4397-4406;Marshall et al. 2006, U.S. Pat. No. 7,037,894), and because no atom isadded in the formation of these bonds, the resulting “staple” can betargeted to be non-disruptive to the protein structure. These tyrosinesmay be present in the primary structure of the protein or added bycontrolled point mutation.

The major advantages of dityrosine crosslinking in protein engineeringinclude (i) the ability to target specific residues for crosslinking(based on the primary, secondary, tertiary, and/or quaternary structuresof proteins and complexes), (ii) minimal structural modification, (iii)specificity of the reaction (tyrosine is the only amino acid known toform crosslinks under specific crosslinking conditions; proteins remainotherwise intact); (iv) stability of the linkage, (v) zero lengthcrosslink (no atom added), and (vi) scalable chemistry.

In some embodiments dityrosine (“DT”) bonds/crosslinks may be targetedto specific residue pairs within the structure of a protein or proteincomplex where DT bonds will, or are predicted to, form, due to, forexample, their close proximity. This may either be done with proteins inwhich, at the targeted residue pair(s) tyrosyl sidechains are alreadypresent, and other tyrosyl side chains will not, or are not predictedto, form DT bonds because, for example, they are not in close enoughproximity to each other. Proteins/protein complexes can also beengineered in such a way that at the targeted residue pair(s), tyrosylsidechains are present, and at residues where it may be undesirable forDT crosslinks to form, at least one of the tyrosyl side chains isreplaced with another side chain, such as a phenylalanine side chain(see, for example, Marshall C P et al., U.S. patent application Ser. No.09/837,235, the contents of which are hereby incorporated by reference).This may be achieved, for example, by introducing point mutations totyrosine or from tyrosine in the nucleic acid sequences directing theexpression of the proteins or protein complexes using any suitablemethods known in the art. Alternatively, the proteins/protein complexesmay be synthesized, purified, and/or produced by any suitable methodsknown in the art to include desirable tyrosine residues and removeundesirable tyrosine residues.

In order to form DT bonds, proteins with tyrosyl side chains at thetargeted residue pair(s) can be subjected to reaction conditions thatlead to the formation of DT bonds. Such conditions are, or become,oxidative reaction conditions, as the DT bond formation reaction is anoxidative crosslink. In some embodiments the DT cross-linking reactionconditions yield proteins that are otherwise not, or not detectably,modified. Such conditions may be obtained by use of enzymes thatcatalyze the formation of H₂O₂, such as peroxidases. DT bond formationmay be monitored by spectrophotometry with an excitation wavelength of320 nm, and fluorescence measured at a wavelength of 400 nm, while lossof tyrosyl fluorescence is monitored also monitored by standardprocedures. When loss of tyrosyl florescence is no longer stoicometricwith DT bond formation, the reaction may be stopped by any methods knownto one skilled in the art, such as, for example, by the addition of areducing agent and subsequent cooling (on ice) or freezing of thesample.

Proteins can also be stabilized using heterologous oligomerizationmotifs, such as timerization motifs. Stabilizing oligomeric proteincomplexes by means of evolutionarily developed motifs in nature can beaccomplished by engineering heterologous oliomerization motifs into thestructure of the polpeptides of the complex.

Where viral proteins form trimers that would be more immunogenic iftrimerization were stabilized in particular conformations, heterologoustrimererization motifs can be used to substitute the protein-proteininteraction domains that mediate trimerization of the wild type viralproteins, and that fit the overall structural confinements/constraintsof the viral protein complex. There are a wide variety of trimerizationdomains in natural proteins that can be used for these purposes such as,for example, but not limited to, those described in Habazettl et al.,2009 (Habazettl et al., 2009. NMR Structure of a Monomeric Intermediateon the Evolutionarily Optimized Assembly Pathway of a SmallTrimerization Domain. J. Mol. Biol. pp. null), Kammerer et al., 2005.(Kammerer et al., 2005. A conserved trimerization motif controls thetopology of short coiled coils. Proc Natl Acad Sci USA 102 (39):13891-13896), Innamorati et al., 2006. (Innamorati et al., 2006. Anintracellular role for the Clq-globular domain. Cell signal 18(6):761-770), and Schelling et al., 2007 (Schelling et al., 2007. Thereovirus 6-1 aspartic acid sandwich: A trimerization motif poised forconformational change. Biol Chem 282(15): 11582-11589)

Stabilizing trimeric protein complexes can also be accomplished usingthe GCN4 and T4 fibrinitin motifs (Pancera et al., 2005. SolubleMimetics of Human Immunodeficiency Virus Type 1 Viral Spikes Produced byReplacement of the Native Trimerization Domain with a HeterologousTrimerization Motif: Characterization and Ligand Binding Analysis. JVirol 79(15): 9954-9969; Guthe et al., 2004. Very fast folding andassociation of a trimerization domain from bacteriophage T4 fibritin. J.Mol. Biol. v 337 pp. 905-15; Papanikolopoulou et al., 2008. Creation ofhybrid nanorods from sequences of natural trimeric fibrous proteinsusing the fibritin trimerization motif. Methods Mol Biol 474:15-33).

Heterologous oligomerization motifs may be introduced by any recombinantmethods known to one of ordinary skill in the art in order to stabilizethe protein-protein interactions of proteins and protein complexes ofpresent invention. Such heterologous oligomerization motifs should fitthe structural confinements/constraints of the protein/protein complex,and are likely to yield best results when introduced in such a way thatthe overstructure of the protein/protein complex is otherwise notdistorted. Heterologous oligomerization domains are therefore preferablyintroduced in their most reduced form/structure, and may be introducedin the presence or absence of additional linkers/spacers known to one ofordinary skill in the art that may minimize distortion of the overallprotein complex structure

If the structure and/or immunogenicity of a polypeptide complex iscompromised or altered by a cross-link reaction, maintaining its overallstructure and function can be achieved by controlling the availabilityof amino acid side-chains for the cross-linking reaction. For example,tyrosyl side-chains that are available for the reaction, but that leadto the distortion of the structure of the complex, and that compromisethe immunogenicity/antigenicity of the complex, can be removed bymutating such residues to another amino acid such as, for example,phenylalanine. Furthermore, point mutations may be introduced atpositions where the amino acid side-chains will react with crosslinkingagents or each other, such that the formation of the bond(s) causes themost beneficial outcome. These positions may also be identified asdescribed herein.

To achieve a stabilized protein or protein complex with enhancedimmunogenicity, positions within each protein can be identified at whicha reactive side-chain would be able to form a bond with a reactiveside-chain elsewhere on the protein/complex. Such positions can beselected both with respect toward maintaining or improving upon theimmunogenicity/antigenicity of the protein/complex, and with respecttoward the suitability of the other position involved in the bond. Thepositions to be cross-linked may therefore selected in pairs.

When at a selected residue a reactive side-chain is not already present,a point mutation may be introduced, for example using molecularbiological methods to introduce such a point mutation into the cDNA of anucleic acid directing its expression, such that a reactive side-chainis present and available for the reaction.

Several strategies may be used to target cross-links to specificlocations in a protein or protein complex. Any method known to oneskilled in the art may be used to identify residue pairs of apolypeptide, protein, or protein complex that, when crosslinked, couldprovide a protein or protein complex that is capable of generating aneutralizing response against a viral epitope, and that may lead to theproduction of neutralizing antibodies in vertebrates, mammals, orpreferably humans. Such methods may be based on the selection processesdescribed in Marshall et al. (U.S. Pat. Nos. 7,037,894 and 7,445, 912,the contents of which are hereby incorporated by reference), wherebystabilization of the protein or protein complex may improve upon itsimmunogenic properties. Any computational methods known to one ofordinary skill in the art may also be used to identify positions atwhich crosslinks could stabilize interactions between regions of thesecondary, tertiary, or quaternary structure of a protein or proteincomplex. Furthermore, screening/scanning of residue pairs by any methodsknown to one of ordinary skill in the art may be used to identifypositions at which the crosslink(s) for in the polypeptides, proteins,or protein complexes of the present invention and provide(s) them thecapability of generating neutralizing or broadly neutralizing immuneresponses advantages of the present invention. Any other methods knownto one of ordinary skill in the art may be used, including for example,the use of data matagenic analyses (for example, but not limited to,alanine screening).

Where proteins or protein complexes of the present invention arecross-linked for the purpose of stabilizing one or more particularconformations of a protein, or for the purpose of stabilizingprotein-protein interactions in a protein complex, the chemicalmodifications may be applied by standard methods known to one ofordinary skill in the art, for example after a protein is prepared,expressed, and/or purified. Any one, or a combination of, the targetingstrategies and cross-linking strategies described herein, or known inthe art, may be used. Alternatively, the modification may not betargeted, and proteins with the desired modifications, activities,and/or specificities may be isolated from a mixture of modified andunmodified proteins made using a non-targeted cross-linking system.

The methods and compositions of the present invention can be used inconjunction with proteins and protein complexes from any suitable virus.In some embodiments the viruses are pathogenic viruses. In someembodiments the viruses are enveloped viruses, such as pathogenicenveloped viruses. In some embodiments the viruses are enveloped DNA andRNA viruses, such as, for example, Herpesviruses, includingAlphaherpesvirinee, Betaherpesvirinae, Gammaherpesvirinae, Simplexvirus,Human herpesvirus 1, Varicellovirus, Human herpesvirus 3 (orVaricella-zoster virus), Mardivirus, Gallid herpesvirus 2, Iltovirus,Gallid herpesvirus 1, Cytomegalovirus, Human herpesvirus 5,Muromegalovirus, Murid herpesvirus 1, Roseolovirus, Human herpesvirus 6,Roseolovirus, Human herpesvirus 7, Proboscivirus, Elephantid herpesvirus1, Lymphocryptovirus, Human herpesvirus 4 or Epstein-Barr virusm,Rhadinovirus, Human Herpesvirus 8, Saimiriine herpesvirus 2, Macavirus,Alcelaphine herpesvirus 1, Genus Percavirus, Equid herpesvirus 2,Cercopithecine, and Cercopithecine herpesvirus 1; Poxviruses, includingthe orthopox, parapox, yatapox, molluscipox, variola virus, vacciniavirus, cowpox virus, monkeypox virus, smallpox, orf virus, pseudocowpox,bovine papular stomatitis virus, tanapox virus, yaba monkey tumor virus,and molluscum contagiosum virus; Flaviviruses, including tick- andmosquito-borne, viruses with no known arthropod vector, Gadgets Gullyvirus (GGYV), Kadam virus (KADV), Kyasanur Forest disease virus (KFDV),Langat virus (LGTV), Omsk hemorrhagic fever virus (OHFV), Powassan virus(POWV), Royal Farm virus (RFV), Tick-borne encephalitis virus (TBEV),Louping ill virus (LIV), Meaban virus (MEAV), Saumarez Reef virus(SREV), Tyuleniy virus (TYUV), Aroa virus (AROAV), the Dengue virusgroup, Dengue virus (DENY), Kedougou virus (KEDV), the Japaneseencephalitis virus group, Cacipacore virus (CPCV), Koutango virus(KOUV), Japanese encephalitis virus (JEV), Murray Valley encephalitisvirus (MVEV), St. Louis encephalitis virus (SLEV), Usutu virus (USUV),West Nile virus (WNV), Yaounde virus (YAOV), Kokobera virus (KOKV), theNtaya virus group, Bagaza virus (BAGV), Ilheus virus (ILHV), Israelturkey meningoencephalomyelitis virus (ITV), Ntaya virus (NTAV), Tembusuvirus (TMUV), the Spondweni virus group, Zika virus (ZIKV), the Yellowfever virus group, Banzi virus (BANV), Bouboui virus (BOUV), Edge Hillvirus (EHV), Jugra virus (JUGV), Saboya virus (SABV), Sepik virus(SEPV), Uganda S virus (UGSV), Wesselsbron virus (WESSV), Yellow fevervirus (YFV), the Entebbe virus group, Entebbe bat virus (ENTV), Yokosevirus (YOKV), the Modoc virus group, Apoi virus (APOIV), Cowbone Ridgevirus (CRV), Jutiapa virus (JUTV), Modoc virus (MODV), Sal Vieja virus(SVV), San Perlita virus (SPV), the Rio Bravo virus group, Bukalasa batvirus (BBV), Carey Island virus (CIV), Dakar bat virus (DBV), Montanamyotis leukoencephalitis virus (MMLV), Phnom Penh bat virus (PPBV), andthe Rio Bravo virus (RBV); Togaviruses, including Alphavirus, Rubivirus,Sindbis virus, Eastern equine encephalitis virus, Western equineencephalitis virus, Venezuelan equine encephalitis virus, Ross Rivervirus, O'nyong'nyong virus, and Rubella virus; Coronaviruses, includingGroup 1, Group 2, Group 3, Canine coronavirus (CCoV), Feline coronavirus(FeCoV), Human coronavirus 229E (HCoV-229E), Porcine epidemic diarrheavirus (PEDV), Transmissible gastroenteritis virus (TGEV), HumanCoronavirus NL63 (NL or New Haven), Bovine coronavirus (BCoV), Caninerespiratory coronavirus (CRCoV), Human coronavirus OC43 (HCoV-OC43),Mouse hepatitis virus (MHV), Porcine hemagglutinating encephalomyelitisvirus (HEV), Rat coronavirus (RCV) Turkey coronavirus (TCoV), HCoV-HKU1,Infectious bronchitis virus (IBV), Turkey coronavirus (Bluecomb diseasevirus), and Severe acute respiratory syndrome coronavirus (SARS-CoV);Hepatitis D virus; Orthomysoviruses, including influenza A, B, and Cviruses, Infectious salmon anemia virus, and Thogotovirus;Mononegavirales; Paramyxoviruses, including the Paramyxovirinae,Pneumovirinae, Newcastle disease virus, Hendravirus, Nipahvirus, Measlesvirus, Rinderpest virus, Canine distemper virus, phocine distempervirus, Peste des Petits Ruminants virus (PPR), Sendai virus, Humanparainfluenza viruses 1 and 3, some of the viruses of the common cold,Mumps virus, Simian parainfluenza virus 5, Menangle virus, Tioman virus,Tupaia paramyxovirus, Human respiratory syncytial virus, Bovinerespiratory syncytial virus, Avian pneumovirus, Human metapneumovirus,Fer-de-Lance virus, Nariva virus, Tupaia paramyxovirus, Salem virus, Jvirus, Mossman virus, and Beilong virus; Rhabdoviruses, including theVesicular stomatitis Indiana virus, Rabies virus, Bovine ephemeral fevervirus, and Infectious haematopoetic necrosis virus; Bunyaviruses,including the Hantavirus; type species, Dugbe virus, Bunyamwera virus,Rift Valley fever virus, and Tenuivirus; Filoviruses, including fivesubtypes of the Ebola virus and the Marburg virus (Marburgvirus);Reoviruses, including Turreted and Nonturreted Reoviruses, AquareovirusA, Cypovirus 1 (CPV 1), Fiji disease virus, Idnoreovirus 1, Mycoreovirus1, Mammalian orthoreovirus, Colorado tick fever virus (CTFV), Bluetonguevirus, Rotavirus A, and Seadornavirus; Hepadnaviruses, includingHepatitis B virus and Duck hepatitis B virus; and Retroviruses,including the Avian leukosis virus, Mouse mammary tumour virus, Murineleukemia virus, Feline leukemia virus, Bovine leukemia virus, HumanT-lymphotropic virus, Walleye dermal sarcoma virus, Chimpanzee foamyvirus, the Lentiviruses, the Simian and Feline immunodeficiency virus,the Human Immunodeficiency Virus Type 1, Group M and Subtypes A, B, C,D, E, F, G, H, I, J, and K, Group N, and Group 0, and HumanImmunodeficiency Virus Type 2; and any groups, subgroups, families,subfamilies, types, subtypes, genuses, species, strains, and/or cladesof the any of the foregoing.

Diseases that may be caused by, or be associated with infection by, suchpathogenic enveloped viruses include, but are not limited to, AIDS,Alzheimer's disease, atherosclerosis, bovine diarrhea, bovine ephemeralfever, bovine papular stomatitis, bronchiolitis, bronchitis, Burkitt'slymphoma, canine distemper, cold sores, chickenpox, chikungunya virusdisease, cholangio carcinoma, chronic fatigue syndrome, the common cold,cowpox, Crohn's disease, diarrhea, dysautomnia, Dengue fever,encephalitis (in human and animal, e.g. equine), exanthem subitum,fibromyalgia, gastroenteritis, genital herpes, hantavirus pulmonarysyndrome, hendra virus disease (haemorrhage and oedema of the lungs, andmeningitis), hepatitis, hepatocarcinoma, Hodgkin's disease, infectioushaematopoetic necrosis, infectious salmon anemia virus, influenza,Korean hemorrhagic fever, measles, mononucleosis, multiple sclerosis,mumps, Newcastle disease, nasopharyngeal carcinoma, pancreatic cancer,pancreatitis, pityriasis rosea, pneumonia, porcine transmissiblegastroenteritis, rabies, respiratory tract infections (upper and lowerrespiratory tract), rinderpest, roseola infantum, shingles, small pox,vesicular stomatitis Indiana, viral hemorrhagic fevers, and west nilefever.

Nucleic acids encoding proteins/complexes of the present invention areprovided. The proteins/complexes can be made by expressing nucleic acidsequences that encode them in vitro or in vivo by any known method knownto one of ordinary skill in the art. Nucleic acids encodingproteins/complexes can be made by altering nucleic acid sequencesencoding proteins/complexes by, for example, substitutions, additions(e.g., insertions) or deletions. The sequences can be cleaved atappropriate sites with restriction endonuclease(s), followed by furtherenzymatic modification if desired, isolated, and ligated in vivo or invitro. Additionally, a nucleic acid sequence can be mutated in vitro orin vivo, to create and/or destroy translation, initiation, and/ortermination sequences, or to create variations in coding regions and/orto form new, or destroy preexisting, restriction endonuclease sites tofacilitate further in vitro modification.

Due to the degeneracy of nucleotide coding sequences, many differentnucleic acid sequences can encode substantially the same residues in aprotein/complex of the present invention. These can include nucleotidesequences comprising all, or portions of, a domain which is altered bythe substitution of different codons that encode the same amino acid, ora functionally equivalent amino acid residue within the sequence, thusproducing a “silent” (or functionally or phenotypically irrelevant)change, or a different amino acid residue with the sequence, thusproducing a functionally or immunoglogically more beneficial change.

Any technique for mutagenesis known to one of ordinary skill in the artcan be used, including but not limited to, enzymatic and chemicalmutagenesis, in vitro site-directed mutagenesis, using, for example, theQuikChange Site-Directed Mutagenesis Kit (Stratagene), etc.

Any prokaryotic or eukaryotic cell can serve as the nucleic acid sourcefor molecular cloning. A nucleic acid sequence encoding a protein ordomain to be engineered for enhanced immunogenicity may be isolated fromsources including prokaryotic, eukaryotic, mono-cellular,multi-cellular, animal, plant, fungus, vertebrate, mammalian, human,porcine, bovine, feline, equine, canine, avian, etc.

The nucleic acid may be obtained by any procedures known to one ofordinary skill in the art, for example, but not limited to, from clonedDNA (e.g., a DNA “library”), by chemical synthesis, by cDNA cloning, bythe cloning of genomic DNA, or fragments thereof, e.g. purified from thedesired cell (see e.g., Sambrook et al., 1985. Glover (ed.). MRL Press,Ltd., Oxford, U.K.; vol. I, II). The nucleic acid may also be obtainedby reverse transcribing cellular RNA, prepared by any of the methodsknown to one of ordinary skill in the art, such as random- or polyA-primed reverse transcription. Such nucleic acid may be amplified usingany of the methods known to one of ordinary skill in the art, includingPCR and 5′ RACE techniques (Weis J. H. et al., 1992. Trends Genet. 8(8):263-4; Frohman M A, 1994. PCR Methods Appl. 4(1): S40-58).

Whatever the source, the nucleic acid can be molecularly cloned into asuitable vector for propagation of the nucleic acid. Additionally, thenucleic acid may be cleaved at specific sites using various restrictionenzymes, DNAse may be used in the presence of manganese, or the DNA canbe physically sheared, as for example, by sonication. The linear DNAfragments can then be separated according to size by standardtechniques, such as agarose and polyacrylamide gel electrophoresis andcolumn chromatography.

Once nucleic acid fragments are generated, identification of specificnucleic acid fragments containing the desired sequences may beaccomplished by any method known to one of ordinary skill in the art. Asnon-limiting examples, clones can be isolated by using PCR techniquesthat may either use two oligonucleotides specific for the desiredsequence, or a single oligonucleotide specific for the desired sequence,using, for example, the 5′ RACE system (Cale J M et al., 1998. MethodsMol. Biol. 105: 351-71; Frohman M A, 1994. PCR Methods Appl. 4(1):S40-58). The oligonucleotides may or may not contain degeneratenucleotide residues. Alternatively, if a portion of a nucleic acid isavailable and can be purified and labeled to generate a probe fornucleic acid hybridization (e.g. Benton and Davis, 1977. Science196(4286): 180-2). Nucleic acid sequences with substantial homology tothe probe will hybridize to it and can be detected and isolated. It mayalso be possible to identify the appropriate fragment by restrictionenzyme digestion(s) and comparison of fragment sizes with those expectedaccording to a known restriction map if such is available. Furtherselection can be carried out on the basis of the properties of the gene.

The presence of a desired nucleic acid may also be detected by assaysbased on the physical, chemical, or immunological properties of itsexpressed product. For example, cDNA clones, or DNA clones whichhybrid-select the proper mRNAs, can be selected and expressed to producea protein that has, for example, similar or identical electrophoreticmigration, isoelectric focusing behavior, proteolytic digestion maps,hormonal or other biological activity, binding activity, or antigenicproperties as known for a protein.

Using an antibody to a known protein, other proteins may be identifiedby binding of the labeled antibody to expressed putative proteins, forexample, in an ELISA (enzyme-linked immunosorbent assay)-type procedure.Further, using a binding protein specific to a known protein, otherproteins may be identified by binding to such a protein either in vitroor a suitable cell system, such as the yeast-two-hybrid system (see e.g.Clemmons D R, 1993. Mol. Reprod. Dev. 35: 368-74; Loddick S A, 1998 etal. Proc. Natl. Acad. Sci., U.S.A. 95:1894-98).

A gene can also be identified by mRNA selection using nucleic acidhybridization followed by in vitro translation. In this procedure,fragments are used to isolate complementary mRNAs by hybridization. SuchDNA fragments may represent available, purified DNA of another species(e.g., Drosophila, mouse, human). Immunoprecipitation analysis orfunctional assays (e.g. aggregation ability in vitro, binding toreceptor, etc.) of the in vitro translation products of the isolatedproducts of the isolated mRNAs identifies the mRNA and, therefore, thecomplementary DNA fragments that contain the desired sequences.

In addition, specific mRNAs may be selected by adsorption of polysomesisolated from cells to immobilized antibodies specifically directedagainst protein. A radiolabeled cDNA can be synthesized using theselected mRNA (from the adsorbed polysomes) as a template. Theradiolabeled mRNA or cDNA may then be used as a probe to identify theDNA fragments from among other genomic DNA fragments.

Alternatives to isolating the genomic nucleic acid sequences encoding aprotein include chemically synthesizing the nucleic acid sequences ormaking cDNA from an mRNA which encodes the protein. For example, RNA foruse in cDNA cloning of a nucleic acid encoding a protein of interest canbe isolated from cells that express that protein.

The identified and isolated nucleic acid can be inserted into anyappropriate cloning or expression vector known to one of ordinary skillin the art. A large number of vector-host systems known in the art maybe used. Possible vectors include plasmids or modified viruses, but thevector system must be compatible with the host cell used. Such vectorsinclude bacteriophages such as lambda derivatives, or plasmids such asPBR322 or pUC plasmid derivatives or the Bluescript vector (Stratagene).

The insertion into a cloning vector can, for example, be accomplished byligating the DNA fragment into a cloning vector that has complementarycohesive termini. However, if the complementary restriction sites usedto fragment the DNA are not present in the cloning vector, the ends ofthe DNA molecules may be enzymatically modified. Alternatively, any sitedesired may be produced by ligating nucleotide sequences (linkers) ontothe DNA termini; these ligated linkers may comprise specific chemicallysynthesized oligonucleotides encoding restriction endonucleaserecognition sequences. Furthermore, the gene and/or the vector may beamplified using PCR techniques and oligonucleotides specific for thetermini of the gene and/or the vector that contain additionalnucleotides that provide the desired complementary cohesive termini. Inalternative methods, the cleaved vector and a gene may be modified byhomopolymeric tailing (Cale J M et al., 1998. Methods Mol. Biol. 105:351-71). Recombinant molecules can be introduced into host cells viatransformation, transfection, infection, electroporation, etc., so thatmany copies of the gene sequence are generated.

In specific embodiments, transformation of host cells with recombinantDNA molecules that incorporate an isolated gene, cDNA, or synthesizedDNA sequence enables generation of multiple copies of the gene. Thus,the gene may be obtained in large quantities by growing transformants,isolating the recombinant DNA molecules from the transformants and, whennecessary, retrieving the inserted gene from the isolated recombinantDNA.

The sequences provided by the present invention include those nucleotidesequences encoding substantially the same amino acid sequences as foundin native proteins, and those encoded amino acid sequences withfunctionally equivalent amino acids, as well as those encoding otherderivatives or analogs, as described below for derivatives and analogs.

The amino acid sequence of a protein may be derived by any method knownto one of ordinary skill in the art. For example, the sequence can bederived by deduction from the DNA sequence, or alternatively, by directsequencing of the protein, for example, with an automated amino acidsequencer.

A protein sequence may be further characterized by any method known toone of ordinary skill in the art. For example, a protein can becharacterized by a hydrophilicity analysis (Hopp T P & Woods K R, 1981.Proc. Natl. Acad. Sci., U.S.A. 78: 3824). A hydrophilicity profile canbe used to identify the hydrophobic and hydrophilic regions of theprotein and the corresponding regions of the gene sequence, which encodesuch regions.

Secondary, structural analysis may be carried out by any method known toone of ordinary skill in the art (e.g. Chou P Y & Fasman G D, 1974.Biochemistry 13(2): 222-45). For example, secondary structural analysiscan also be done, to identify regions of a protein that assume specificsecondary structures. Manipulation, translation, and secondary structureprediction, open reading frame prediction and plotting, as well asdetermination of sequence homologies, can also be accomplished usingcomputer software programs available in the art. Other methods ofstructural analysis include X-ray crystallography, nuclear magneticresonance spectroscopy and computer modeling.

The nucleotide sequence coding for a protein/complex can be insertedinto an appropriate expansion or expression vectors, i.e., a vectorwhich contains the necessary elements for the transcription alone, ortranscription and translation, of the inserted protein-codingsequence(s). The native genes and/or their flanking sequences can alsosupply the necessary transcriptional and/or translational signals.

Expression of a nucleic acid sequence encoding a protein or proteincomplex may be regulated by a second nucleic acid sequence so that thepolypeptide is expressed in a host transformed with the recombinant DNAmolecule. For example, expression of a polypeptide may be controlled byany promoter/enhancer element known in the art.

Promoters which may be used to control gene expression include, asexamples, the SV40 early promoter region, the promoter contained in the3′ long terminal repeat of Rous sarcoma, the herpes thymidine kinasepromoter, the regulatory sequences of the metallothionein gene;prokaryotic expression vectors such as the β-lactamase promoter, or thelac promoter; plant expression vectors comprising the nopalinesynthetase promoter or the cauliflower mosaic virus 35S RNA promoter,and the promoter of the photosynthetic enzyme ribulose biphosphatecarboxylase; promoter elements from yeast or other fungi such as the Gal4 promoter, the alcohol dehydrogenase promoter, phosphoglycerol kinasepromoter, alkaline phosphatase promoter, and the following animaltranscriptional control regions, which exhibit tissue specificity andhave been utilized in transgenic animals: elastase I gene control regionwhich is active in pancreatic acinar cells (Swift et al. Cell; vol. 38:pp. 639-646, 1984); a gene control region which is active in pancreaticbeta cells (Hanahan D., Nature; vol. 315: pp. 115-122, 1985), animmunoglobulin gene control region which is active in lymphoid cells(Grosschedl R. et al. Cell; vol. 38: pp. 647-658, 1984), mouse mammarytumor virus control region which is active in testicular, breast,lymphoid and mast cells (Leder A. et al. Cell; vol. 45: pp. 45-495,1986), albumin gene control region which is active in liver (Pinkert C.A. et al. Genes Dev.; vol. 1: pp. 268-276, 1987), alpha-fetoprotein genecontrol region which is active in liver (Krumlauf R. et al. Mol. Cell.Biol.; vol. 5: pp. 1639-1648, 1985); alpha 1-antitrypsin gene controlregion which is active in the liver (Kelsey G. D. et al. Genes Dev.;vol. 1: pp. 161-171, 1987), beta-globin gene control region which isactive in myeloid cells (Magram J. et al. Nature; vol. 315: pp. 338-340,1985); myelin basic protein gene control region which is active inoligodendrocyte cells in the brain (Readhead C. et al. Cell; vol. 48:pp. 703-712, 1987); myosin light chain-2 gene control region which isactive in skeletal muscle (Shani M. Nature; vol. 314: pp. 283-286,1985), and gonadotropic releasing hormone gene control region which isactive in the hypothalamus (Mason A. J. et al. Science; vol. 234: pp.1372-1378, 1986).

In some embodiments a vector is used that comprises a promoter operablylinked to a nucleic acid, one or more origins of replication, and,optionally, one or more selectable markers (e.g., an antibioticresistance gene). In bacteria, the expression system may comprise thelac-response system for selection of bacteria that contain the vector.Expression constructs can be made, for example, by subcloning a codingsequence into one the restriction sites of each or any of the pGEXvectors (Pharmacia, Smith D. B. and Johnson K. S. Gene; vol. 67: pp.31-40, 1988). This allows for the expression of the protein product.

Vectors containing nucleic acid inserts can be identified by severaldifferent approaches, including: (a) identification of specific one orseveral attributes of the nucleic acid itself, such as, for example,fragment lengths yielded by restriction endonuclease treatment, directsequencing, PCR, or nucleic acid hybridization; (b) presence or absenceof “marker” functions; and, where the vector is an expression vector,(c) expression of inserted sequences. In the first approach, thepresence of a gene inserted in a vector can be detected, for example, bysequencing, PCR or nucleic acid hybridization using probes comprisingsequences that are homologous to an inserted gene. In the secondapproach, the recombinant vector/host system can be identified andselected based upon the presence or absence of certain “marker” genefunctions (e.g., thymidine kinase activity, resistance to antibiotics,transformation phenotype, occlusion body formation in baculovirus, etc.)caused by the insertion of a gene in the vector. For example, if thenucleic acid is inserted within the marker gene sequence of the vector,recombinants containing the insert an identified by the absence of themarker gene function. In the third approach, recombinant expressionvectors can be identified by assaying the product expressed by therecombinant expression vectors containing the inserted sequences. Suchassays can be based, for example, on the physical or functionalproperties of the protein in in vitro assay systems, for example,binding with anti-protein antibody.

Once a particular recombinant nucleic acid molecule is identified andisolated, several methods known in the art may be used to propagate it.Once a suitable host system and growth conditions are established,recombinant expression vectors can be propagated and prepared inquantity. Some of the expression vectors that can be used include humanor animal viruses such as vaccinia virus or adenovirus; insect virusessuch as baculovirus; yeast vectors; bacteriophage vectors (e.g., lambdaphage), and plasmid and cosmid DNA vectors.

Once a recombinant vector that directs the expression of a desiredsequence is identified, the gene product can be analyzed. This isachieved by assays based on the physical or functional properties of theproduct, including radioactive labeling of the product followed byanalysis by gel electrophoresis, immunoassay, etc.

A variety of host-vector systems may be utilized to express theprotein-coding sequences. These include, as examples, mammalian cellsystems infected with virus (e.g., vaccinia virus, adenovirus, etc.);insect cell systems infected with virus (e.g., baculovirus);microorganisms such as yeast containing yeast vectors, or bacteriatransformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. Theexpression elements of vectors vary in their strengths andspecificities. Depending on the host-vector system utilized, any one ofa number of suitable transcription and translation elements may be used.

In some embodiments the gene may be expressed in bacteria that areprotease deficient, and that have low constitutive levels and highinduced levels of expression where an expression vector is used that isinducible, for example, by the addition of IPTG to the medium.

In yet another embodiment, the proteins/complexes may be expressed withsignal peptides, such as, for example, pelB bacterial signal peptide,that directs the protein to the bacterial periplasm (Lei et al. J.Bacteril., vol. 169: pp. 4379, 1987). Alternatively, protein may beallowed to form inclusion bodies, and subsequently be resolubilzed andrefolded (Kim S. H. et al. Mo Immunol, vol. 34: pp. 891, 1997).

In yet another embodiment, a fragment of one, any, both, several or allof the proteins a complex comprising one or more domains of the proteinis expressed. Any of the methods previously described for the insertionof DNA fragments into a vector may be used to construct expressionvectors containing a chimeric gene consisting of appropriatetranscriptional/translational control signals and the protein codingsequences. These methods may include in vitro recombinant DNA andsynthetic techniques and in vivo recombinants (genetic recombination).

In addition, a host cell strain may be chosen that modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Expression from certainpromoters can be elevated in the presence of certain inducers; thus,expression of the genetically engineered polypeptides may be controlled.Furthermore, different host cells have characteristic and specificmechanisms for the translational and post-translational processing andmodification (e.g., glycosylation, phosphorylation of proteins.Appropriate cell lines or host systems can be chosen to ensure thedesired modification and processing of the foreign polypeptide(s)expressed. For example, expression in a bacterial system can be used toproduce a non-glycosylated core protein product. Expression in yeastwill produce a glycosylated product. Expression in mammalian cells canbe used to ensure “native” glycosylation of a heterologous protein.Furthermore, different vector/host expression systems may effectprocessing reactions to different extents.

In other embodiments of the invention, proteins and complexes of thepresent invention, and any derivates, analogs, orthologs, homologs, orfragments thereof, and one, any, both, several or all of thepolypeptides a complex, and any derivates, analogs, orthologs, homologs,or fragments thereof may be expressed as a fusion-, or chimeric, proteinproduct (comprising the protein, fragment, analog, or derivative joinedvia a peptide bond to a heterologous protein sequence of a differentprotein). Such a chimeric product can be made by ligating theappropriate nucleic acid sequences encoding the desired amino acidsequences to each other by methods known in the art, in the propercoding frame, and expressing the chimeric product by methods commonlyknown in the art. Alternatively, such a chimeric product may be made byprotein synthetic techniques, for example, by use of a peptidesynthesizer.

The proteins and protein complexes may be expressed together in the samecells either on the same vector, driven by the same or independenttranscriptional and/or translational signals, or on separate expressionvectors, for example by cotransfection or cotransformation andselection, for example, may be based on both vectors' individualselection markers. Alternatively, proteins/complexes may be expressedseparately; they may be expressed in the same expression system, or indifferent expression systems, and may be expressed individually orcollectively as fragments, derivatives or analogs of the originalpolypeptide.

Any method known to one of ordinary skill in the art may be used toidentify epitopes of polypeptides and one, any, both, several or all ofthe polypeptides a complex, and any derivates, analogs, orthologs,homologs, fragments, chimers, or fusion proteins thereof, that areimmunogenic, and that lead to the production of neutralizing and/orbroadly neutralizing antibodies. Such methods may, as non-limitingexamples, be computational or based on antigenic studies usingantibodies known to be neutralizing and/or broadly neutralizing or usingneutralizing and/or broadly neutralizing antibodies that result fromimmunization with polypeptides and one, any, both, several or all of thepolypeptides a complex, and any derivates, analogs, orthologs, homologs,fragments, chimers, or fusion proteins thereof.

Antigenic analyses, i.e. the determination of whether the engineeredpolypeptides and one, any, both, several or all of the polypeptides acomplex, and any derivates, analogs, orthologs, homologs, fragments,chimers, or fusion proteins thereof, bind specific antibodies known tobind to any, or to specific antigenic structures of a particularconformation of a polypeptide or protein of the present invention, orany derivate, analog, ortholog, homolog, fragment, chimer, or fusionprotein thereof, and one, any, both, several or all of the polypeptidesa complex, and any derivates, analogs, orthologs, homologs, fragments,chimers, or fusion proteins thereof, may be performed by any methodknown in the art. Such methods include, as non-limiting examples, thosedescribed in detail by Dey et al. 2007 (Dey et al., 2007.Characterization of Human Immunodeficiency Virus Type 1 Monomeric andTrimeric gp120 Glycoproteins Stabilized in the CD4-Bound State:Antigenicity, Biophysics, and Immunogenicity. J Virol 81(11):5579-5593), Binley et al., 2000 (Binley et al., 2000. A RecombinantHuman Immunodeficiency Virus Type 1 Envelope Glycoprotein ComplexStabilized by an Intermolecular Disulfide Bond between the gp120 andgp41 Subunits Is an Antigenic Mimic of the Trimeric Virion-AssociatedStructure. J Virol 74(2): 627-643), Pancera et al., 2005 (Pancera etal., 2005. Soluble Mimetics of Human Immunodeficiency Virus Type 1 ViralSpikes Produced by Replacement of the Native Trimerization Domain with aHeterologous Trimerization Motif Characterization and Ligand BindingAnalysis. J Virol 79(15): 9954-9969), and Beddows et al., 2006 (Beddowset al., 2006. Construction and Characterization of Suluble, Cleaved, andStabilized Trimeric Env proteins Based on HIV Type 1 env Subtype A. AIDSRes Hum Retroviruses 22(6): 569-579).

Immunogenic analyses, i.e. the determination of whether the engineeredpolypeptides and one, any, both, several or all of the polypeptides acomplex, and any derivates, analogs, orthologs, homologs, fragments,chimers, or fusion proteins thereof, used as immunogens generateantibodies known to bind to specific antigenic structures of any, or ofany particular conformation of a polypeptide or protein of the presentinvention, or any derivate, analog, ortholog, homolog, fragment, chimer,or fusion protein thereof, and one, any, both, several or all of thepolypeptides a complex, and any derivates, analogs, orthologs, homologs,fragments, chimers, or fusion proteins thereof, may be performed by anymethod known in the art. Such methods include, as non-limiting examples,those described in detail by Dey et al. 2007 (Dey et al., 2007.Characterization of Human Immunodeficiency Virus Type 1 Monomeric andTrimeric gp120 Glycoproteins Stabilized in the CD4-Bound State:Antigenicity, Biophysics, and Immunogenicity. J Virol 81(11): 5579-5593)and Beddows et al., 2006 (Beddows et al., 2007. A comparativeimmunogenicity study in rabbits of disulfide-stabilized proteolyticallycleaved, soluble trimeric human immunodeficiency virus type 1 gp140,trimeric cleavage-defective gp140 and momomeric gp120. Virol 360:329-340).

Neutralization assays, i.e. the determination of whether antibodies orantisera generated by immunization of vertebrates, preferably mammals,such as, for example, but not limited to mice, rabbits, or primates,with the engineered polypeptides and one, any, both, several or all ofthe polypeptides a complex, and any derivates, analogs, orthologs,homologs, fragments, chimers, or fusion proteins thereof, have viralneutralizing activity, may be performed by any method known in the art.Such methods include, as non-limiting examples, those described indetail by Dey et al. 2007 (Dey et al., 2007. Characterization of HumanImmunodeficiency Virus Type 1 Monomeric and Trimeric gp120 GlycoproteinsStabilized in the CD4-Bound State: Antigenicity, Biophysics, andImmunogenicity. J Virol 81(11): 5579-5593) and Beddows et al., 2006(Beddows et al., 2007. A comparative immunogenicity study in rabbits ofdisulfide-stabilized proteolytically cleaved, soluble trimeric humanimmunodeficiency virus type 1 gp140, trimeric cleavage-defective gp140and momomeric gp120. Virol 360: 329-340).

Biophysical analyses, i.e. the determination of any biophysicalcharacteristics known in the art, such as, for example, but not limitedto, stability of engineered polypeptides and of one, any, both, severalor all of the polypeptides a complex, and any derivates, analogs,orthologs, homologs, fragments, chimers, or fusion proteins thereof, andof the complex itself, may be performed by any method known in the art.

Stability of the engineered material may be tested in vitro in, asexamples, but not limited to, denaturing and non-denaturingelectrophoresis by any methods known to one of ordinary skill in theart, by isothermal titration calorimetry, as described in detail in Deyet al., 2007 ((Dey et al., 2007. Characterization of HumanImmunodeficiency Virus Type 1 Monomeric and Trimeric gp120 GlycoproteinsStabilized in the CD4-Bound State: Antigenicity, Biophysics, andImmunogenicity. J Virol 81(11): 5579-5593), and time-course experimentsincubating the polypeptides and one, any, both, several or all of thepolypeptides a complex, and any derivates, analogs, orthologs, homologs,fragments, chimers, or fusion proteins thereof, at varying proteinconcentrations and temperatures; the engineered material's stability mayalso be tested at various pH levels and in various redox conditions. Forthe above conditions, as non-limiting examples, the antigenicity,immunogenicity, and neutralization capacity of the polypeptides and one,any, both, several or all of the polypeptides a complex, and anyderivates, analogs, orthologs, homologs, fragments, chimers, or fusionproteins thereof, are determined by assaying as described above.Proteins may be incubated at varying temperatures in serum, or otherbiologically derived media, and may be analyzed for susceptibility toproteolytic degradation by any methods known to one of ordinary skill inthe art.

To determine the utility of an engineered polypeptide, protein, orprotein complex more directly, biodistribution and/or otherpharmacokinetic attributes may be determined. In a specific embodimentengineered material may be injected into a model organism and assayedfor by tracing a marker, such as, for example, but not limited to, ¹²⁵Ior ¹⁸F radio labels (Choi C W et al, 1995. Cancer Research 55: 5323-29),and/or by tracing activity as described above (Colcher D et al., 1998.Q. J. Nucl. Med. 44(4): 225-41). Relevant information may be obtained,for example, by determining the amount of material that can be expectedto be immunogenically active due to its penetration of the targetedtissue. Half-life in circulation and at the targeted tissue, clearance,immunogenicity, and speed of penetration may also be determined in thiscontext.

The most conclusive measurements with regard to a conjugate's utility asa vaccine immunogen are to determine its immunogenic activity directlyclinically. In a specific embodiment, such studies may assess, forexample, but not limited to, the level of protection afforded byengineered polypeptides and one, any, both, several or all of thepolypeptides a complex, and any derivates, analogs, orthologs, homologs,fragments, chimers, or fusion proteins thereof. For example, acomparison may be made between placebo and immunogen vaccinated groupswith regard to their rates of infection (or sero-conversion). As anothernon-limiting example, the therapeutic capacity of the engineeredpolypeptides and one, any, both, several or all of the polypeptides acomplex, and any derivates, analogs, orthologs, homologs, fragments,chimers, or fusion proteins thereof, may be assesses. For example, acomparison may be made between placebo and immunogen vaccinated groupswith regard to their viral loads, or, in the case of an HIV vaccine, asa non-limiting example, with regard CD4 cell counts.

This invention provides software that permits automated selection ofsuitable residues at which a polypeptide, protein, or protein complexmay be modified for crosslinking. Such software can be used inaccordance with the selection process, as described above, and withgeometrical, physical, and chemical criteria, such as set forth in theUS patent “Stabilized Proteins” (Marshall C P et al., U.S. Pat. No.7,445,912; see especially Identification of Suitable Residue Pairs forthe Reaction, Software for the Residue Selection Process in Section 5,and the Residue Pair Selection Flowchart in Section 6).

In some embodiments the present invention is directed to pharmaceuticalcompositions, and administration of such pharmaceutical compositions tosubjects. In some embodiments the subjects are animal species. In somethe subjects are mammalian animal species. In some embodiments thesubject are humans. In some embodiments the pharmaceutical compositionsof the invention may comprise, or consist essentially of, the engineeredproteins and protein complexes described herein. In some embodiments theengineered proteins or protein complexes of the present invention may beprovided in pharmaceutical composition that comprises one or moreadditional active components, such as one or more additional vaccineimmunogens. In some embodiments the engineered proteins and/or proteincomplexes of the invention may be provided in a pharmaceuticalcomposition that comprises one or more other components, including, butnot limited to, pharmaceutically acceptable carriers, adjuvants, wettingor emulsifying agents, pH buffering agents, preservatives, and/or anyother components suitable for the intended use of the pharmaceuticalcompositions. These pharmaceutical compositions of the invention cantake the form of solutions, suspensions, emulsions and the like. Theterm “pharmaceutically acceptable carrier” includes various diluents,excipients and/or vehicles in which, or with which, the engineeredproteins and protein complexes of the invention can be provided. Theterm “pharmaceutically acceptable carrier” includes, but is not limitedto, carriers known to be safe for delivery to human and/or other animalsubjects, and/or approved by a regulatory agency of the Federal or astate government, and/or listed in the U.S. Pharmacopeia, and/or othergenerally recognized pharmacopeia, and/or receiving specific orindividual approval from one or more generally recognized regulatoryagencies for use in humans and/or other animals. Such pharmaceuticallyacceptable carriers, include, but are not limited to, water, aqueoussolutions (such as saline solutions, buffers, and the like), organicsolvents (such as certain alcohols and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil), and the like. Adjuvants that maybe used include, but are not limited to, inorganic or organic adjuvants,oil-based adjuvants, virosomes, liposomes, lipopolysaccharide (LPS),molecular cages for antigens (such as immune-stimulating complexes(“ISCOMS”)), Ag-modified saponin/cholesterol micelles that form stablecage-like structures that are transported to the draining lymph nodes),components of bacterial cell walls, endocytosed nucleic acids (such asdouble-stranded RNA (dsRNA), single-stranded DNA (ssDNA), andunmethylated CpG dinucleotide-containing DNA), AUM, aluminum phosphate,aluminum hydroxide, and Squalene. In one embodiments virosomes are usedas an adjuvant. Virosomes are known to have an excellent safety profile,and may contain membrane-bound proteins such as hemagglutinin andneuraminidase derived from the influenza virus, which mediate fusogenicactivity and can thereby facilitate uptake of an immunogen (such as theengineered proteins and protein complexes of the invention) by antigenpresenting cells and induce the antigen-processing pathway. Additionalcommercially available adjuvants that can be used in accordance with thepresent invention include, but are not limited to, the Ribi AdjuvantSystem (RAS, an oil-in-water emulsion containing detoxified endotoxin(MPL) and mycobacterial cell wall components in 2% squalene (SigmaM6536)), TiterMax (a stable, metabolizable water-in-oil adjuvant (CytRxCorporation 150 Technology Parkway Technology Park/Atlanta Norcross, Ga.30092)), Syntex Adjuvant Formulation (SAF, an oil-in-water emulsionstabilized by Tween 80 and pluronic polyoxyethlene/polyoxypropyleneblock copolymer L121 (Chiron Corporation, Emeryville, Calif.)), Freund'sComplete Adjuvant, Freund's Incomplete Adjuvant, ALUM—aluminumhydroxide, Al(OH)₃ (available as Alhydrogel, Accurate Chemical &Scientific Co, Westbury, N.Y.), SuperCarrier (Syntex Research 3401Hillview Ave. P.O. Box 10850 Palo Alto, Calif. 94303), Elvax 40W1,2(anethylene-vinyl acetate copolymer (DuPont Chemical Co. Wilmington,Del.)), L-tyrosine co-precipitated with the antigen (available fromnumerous chemical companies); Montanide (a manide-oleate, ISA SeppicFairfield, N.J.)), AdjuPrime (a carbohydrate polymer),Nitrocellulose-absorbed protein, Gerbu adjuvant (C-C Biotech, Poway,Calif.), and the like.

In some embodiments the pharmaceutical compositions of the inventioncomprise an “effective amount” of a protein or protein complex of theinvention. An “effective amount” is an amount required to achieve adesired end result. Examples of desired end results include, but are notlimited to, the generation of a humoral immune response, the generationof a neutralizing antibody response, the generation of a broadlyneutralizing antibody response, and the generation of protectiveimmunity. The amount of an engineered protein or protein complex of theinvention that is effective to achieve the desired end result willdepend on variety of factors including, but not limited to, the natureof the virus against which protection or some other therapeutic effectis sought, the nature of the protein or protein complex, the species ofthe intended subject (e.g. whether a human or some other animalspecies), the age and/or sex of the intended subject, the planned routeof administration, the planned dosing regimen, the seriousness of thedisease or disorder, and the like. The effective amount—which may be arange of effective amounts—can be determined by standard techniqueswithout any undue experimentation, for example using in vitro assaysand/or in vivo assays in the intended subject species or any suitableanimal model species. Suitable assays include, but are not limited to,those that involve extrapolation from dose-response curves and/or otherdata derived from in vitro and/or in vivo model systems. In someembodiments the effective amount may be determined according to thejudgment of a medical or veterinary practitioner based on the specificcircumstances.

Various delivery systems are known in the art and any suitable deliverysystems can be used to administer the pharmaceutical compositions of thepresent invention. Such systems include, but are not limited to,intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous,intranasal, epidural, and oral delivery systems. The pharmaceuticalcompositions may be administered by any convenient route, for example byinfusion or bolus injection, by absorption through epithelial ormucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa,etc.) and may be administered together with other biologically activeagents. Administration can be systemic or local. In addition, it may bedesirable to introduce the pharmaceutical compositions of the inventioninto the central nervous system by any suitable route, includingintraventricular and intrathecal injection. Intraventricular injectionmay be facilitated by an intraventricular catheter, for example,attached to a reservoir, such as an Ommaya reservoir. Pulmonaryadministration can also be employed, e.g., by use of an inhaler ornebulizer, and formulation with an aerosolizing agent.

In some embodiments it may be desirable to administer the pharmaceuticalcompositions of the invention locally to a tissue in which theengineered protein or protein complex may be most effective ingenerating a desirable outcome. This may be achieved by, for example,local infusion, injection, delivery using a catheter, or by means of animplant, such as a porous, non-porous, or gelatinous implant or animplant comprising one or more membranes (such as sialastic membranes)or fibers from or through which the protein or protein complexes may bereleased locally. In some embodiments a controlled release system may beused. In some embodiments a pump may be used (see Langer, supra; Sefton,1987. CRC Crit. Ref. Biomed. Eng. 14: 201; Buchwald et al., 1980.Surgery 88: 507; Saudek et al., 1989. N. Engl. J. Med. 321: 574). Insome embodiments polymeric materials may be used to facilitate and/orcontrol release of the protein or protein complexes of the invention(see Medical Applications of Controlled Release, Langer and Wise (eds.),1974. CRC Pres., Boca Raton, Fla.; Controlled Drug Bioavailability,1984. Drug Product Design and Performance, Smolen and Ball (eds.),Wiley, New York; Ranger & Peppas, 1983 Macromol. Sci. Rev. Macromol.Chem. 23: 61; see also Levy et al., 1985. Science 228:190; During et al,1989. Ann. Neurol. 25: 351; Howard et al., 1989. J. Neurosurg 71:105).In some embodiments a controlled release system can be placed inproximity to the tissue/organ to which the protein or protein complex isto be delivered (see, e.g., Goodson, 1984. in Medical Applications ofControlled Release, supra, vol. 2: 115-138). Some suitable controlledrelease systems that may be used in conjunction with the presentinvention are described Langer, 1990, Science; vol. 249: pp. 527-1533.

EXAMPLES

There has been much focus in HIV vaccine development on engineeringsoluble versions of stabilized HIV envelope (Env) glycoproteins thatrecapitulate properties of the functional Env trimer for use asimmunogens. This approach is taken because gp120 on its own does notefficiently elicit immune responses that generate broadly neutralizingantibodies. Broadly neutralizing antibodies to gp120 have, however, beenisolated from patients.

HIV has evolved several mechanisms of immune evasion inherent in theunmodified HIV envelope glycoproteins. These strategies are based, inpart, on HIV's high rate of mutation, the spike's lability, and thepresence of immuno-dominant variable loops that divert antibodyresponses from functionally conserved epitopes and allow the escape ofviruses with non-cross reactive variable loops. More importantly,however, gp120-receptor interactions involve significant conformationalreorganization, and recognition by antibodies that bind the conservedCD4 receptor binding site (CD4BS) induces conformational change; currenttheory is that the resulting “conformational mask” allows conservedprotein surfaces, such as the CD4BS, to assume various conformations notdisplayed on the functional spike, and enables HIV-1 to maintainfunctionality (receptor binding) while resisting neutralization (Kwonget al. 2002. HIV-1 evades antibody-mediated neutralization throughconformational masking of receptor-binding sites. Nature 420:678-82;Phogat et al, 2007. Rational modifications of HIV-1 envelopeglycoproteins for immunogen design. Curr Pharm Design 13: 213-227). Someprevious studies have suggested that stabilization of envelope proteinsin particular conformations can counteract the conformational maskingstrategy of viruses to evade host immune systems, and stabilize epitopesthat are otherwise poorly or not at all recognized and bound byneutralizing and broadly neutralizing antibodies, and therefore are notsecreted by plasma B cells in response to infection. See, for example,Dey et al., 2007. Characterization of Human Immunodeficiency Virus Type1 Monomeric and Trimeric gp120 Glycoproteins Stabilized in the CD4-BoundState: Antigenicity, Biophysics, and Immunogenicity. J Virol 81(11):5579-5593). Stabilization of the soluble ecto-gp4′-gp120 complex (gp140)by introducing a disulfide bond provides a construct that bindsneutralizing antibodies, but that nonetheless elicits protective humoralimmune responses in animals that are limited in breadth (Beddows et al.,2007. A Comparative Immunogenicity Study in Rabbits ofDisulfide-stabilized, Proteolytically Cleaved, Soluble Trimeric HumanImmunodeficiency Virus Type I gp140, Trimeric Cleavage-Defective gp140and Monomeric gp120. Virology 360: 329-340). Mutations reported tostabilize gp120 in a functionally active conformation allow binding ofthe broadly neutralzing MAb b12 (which binds the conserved CD4BS), andantisera to gp120 stabilized by these mutations demonstrates animprovement in their capacity to neutralize a panel of Glade B viruses(Dey et al., 2007. Characterization of Human Immunodeficiency Virus Type1 Monomeric and Trimeric gp120 Glycoproteins Stabilized in the CD4-BoundState: Antigenicity, Biophysics, and Immunogenicity. J Virol 81(11):5579-5593). More recently, the cryo-electron tomographic structure ofthe trimeric HIV spike has been elucidated at 20 Å resolution, providingstructural information of the spike in unliganded and CD4- andAb-complexed conformations (Liu et al., 2008. Molecular architecture ofnative HIV-1 gp120 trimers. Nature 455: 109). These studies demonstratesthat trimerization is mediated by gp41 and that the three V1/V2 loops ofgp120 come together to form the apex of the spike in the unliganded andb12 and CD4 complexed conformations. Together, these studies suggestthat stabilizing the HIV spike in a particular conformation (which bindsneutralizing antibodies) may be a way to counteract conformationalmasking and obtain an immunogen that elicits broadly protective Abresponses.

Previous studies attempted to stabilize the soluble ecto-gp4′-gp120complex (gp140) of the HIV virus by introducing disulfide bonds, andgenerated a construct that bound to neutralizing antibodies. See Beddowset al., 2007, “A Comparative Immunogenicity Study in Rabbits ofDisulfide-stabilized, Proteolytically Cleaved, Soluble Trimeric HumanImmunodeficiency Virus Type I gp140, Trimeric Cleavage-Defective gp140and Monomeric gp120,” Virology, Vol. 360: pp 329-340. However, disulfidebonds are known to be pH sensitive and to be dissolved under certainredox conditions such that the preventative and/or therapeutic utilityof polypeptides, proteins, or protein complexes engineered withdisulfide crosslinks used as immunogens in vivo may be compromised.Furthermore, undesired disulfide bonds often form between proteins withfree sulfhydryl groups that mediate aggregate formation. As such thereis a need for alternative methods of crosslinking HIV gp120 andecto-gp41 proteins.

Example 1

Stabilization of the protein-protein interactions of the HIV trimericspike via the V1/V2 loop stabilizes the folds of the proteinconformations such that the complex has the capacity to elicitneutralizing or broadly neutralizing humoral immune responses. Liu etal. described the three-dimentional structure of the HIV-1 spike imcomplex with a broadly neutralizing antibody (Liu et al., 2008,Molecular architecture of native HIV-1 gp120 trimers. Nature 455: 109).Based on an analysis of this structure, residues of the V1/V2 loopdistal to the stem between positions 143 and 150, and between positions160 and 180 were selected for formation of dityrosine bonds.

The selected residues are mutated in pair-wise combinations to tyrosine(where tyrosine is not already present), subjected to crosslinkingconditions, whereby the HIV spike is bound and unbound to soluble CD4and the b12 neutralizing antibody, and analyzed for dityrosine bondformation leading to trimerization. Covalent trimerization requires aminimum of two dityrosine bonds to form.

The pcDNA3.1 (Stratagene) vector that is suitable for amplification,mutagenesis, and both stable and transient expression of the HIVproteins and protein complexes. Targeted point mutations in the HIV Envgene are introduced in a pair-wise manner (e.g. 1 in gp120 and 1 ingp41) using the QuikChange Site-Directed Mutagenesis Kit (Stratagene)according to the manufacturer's instructions to generatemutated/engineered proteins. The mutated/engineered proteins areexpressed in serum-free medium by transient transfection of HEK293Tcells. HEK293T cells growing in Dulbecco's modified Eagle's medium with10% fetal bovine serum, 2 mM glutamine, and 1×penicillin-streptomycin(50 units/ml penicillin, 50

strept are seeded at a density of 1.2×10⁷ cells per 150 cm² tissueculture dish, and grown overnight. After the overnight incubation, cellsare transfected with a mixture of the expression vector and thetransfection reagent Fugene (Roche) according to the manufacturer'sinstructions. After approximately 24 hours, the transfection medium isreplaced with 293 SFMII (serum-free medium) supplemented with 4 mMglutamine. After another four days, supernatants are collected, andcentrifugated at 3,500×g. Supernatants are filtered through sterile0.2-□m filters and protease inhibitors are added. Prior to proteinpurification, supernatants can be stored at 4° C. for no more than 1week.

Protein purification is performed using affinity purification columns,and the trimeric fractions are isolated by size exclusionchromatography. An anti-gp120 antibody-coupled affinity column is usedfor the affinity purification. The culture supernatant is applied to theaffinity column overnight at room temperature, and the column is washedwith 10 volumes of phosphate-buffered saline (PBS) (pH 7.4) containing0.5 M NaCl and washed with 5 volumes of PBS containing 0.15MNaCl, andeluted with 100 mM glycine (pH 2.8). The trimeric proteins are elutedwith 3 M MgCl2 prepared in 20 mM Tris-HCl (pH 7.4), and proteinconcentrations are monitored by optical density (OD) at 280 nm. Elutedfractions containing protein are pooled, concentrated with Amicon Ultracentrifugal filter devices (Millipore, Bedford, Mass.), and dialyzedextensively against PBS (pH 7.4) containing protease inhibitors.Affinity-purified trimeric protein is further subjected to sizeexclusion chromatography using a Superdex 200 16/26 column (AmershamPharmacia) in PBS containing 0.35 M NaCl and protease inhibitors. Theflow rate is set to 1 ml/min for the first 100 min and reduced to 0.5ml/min until the end of the run, which allowed the separation of theoligomeric species. Trimeric protein containing fractions are pooled,concentrated as described above, dialyzed against PBS (pH 7.4)containing protease inhibitors, flash-frozen, and stored at −80° C.

For dityrosine crosslinking protein aliquots are subjected to reactionconditions that lead to the formation of dityrosine (DT) bonds incontrol proteins. The reaction is catalyzed enzymatically using theArthromyces Peroxidase as described in detail in Malencik & Anderson,1996, Biochemistry 35: 4375-86. DT bond formation is monitored andquantified by spectrophotometry with an excitation wavelength of 320 nm,and fluorescence measured at a wavelength of 400 nm using a dityrosinestandard (and a standard curve), as described in detail in Malencik &Anderson, 2003, Amino Acids 25: 233-247, while loss of tyrosylfluorescence is monitored also monitored by standard procedures. Whenloss of tyrosyl florescence is no longer stoicometric with DT bondformation, the reaction is stopped by the addition of a reducing agentand subsequent cooling (on ice) or freezing of the sample.

Constructs that are revealed to form dityrosine crosslinks are furtherpurified by standard chromatographic methods, including, for example,size chromatography described above, under mildly denaturing conditionsthat do not cause denaturation, but rather only dissociation ofuncrosslinked monomers. Purified crosslinked constructs are furtheranalyzed, as described below.

Biophysical Analysis Gel Electrophoresis

Standard methods for denaturing and non-denaturing proteins are appliedto confirm, for example, the degree of crosslinking, and to confirm thatthe crosslinking is specifically directed to the targeted tyrosyl sidechains (and that the constructs do not form mulitmers, concatamers,etc.).

Isothermal Titration Calorimetry (ITC)

The degree of thermodynamic stabilization of the dityrosine crosslinkeddimeric complex is quantified by standard ITC methods using sCD4 as aligand and a VP-ITC titration calorimeter system from MicroCal, Inc.Protein samples are dialyzed against PBS and degassed before use. Theenvelope protein concentration in the sample cell is approximately 4 μM,and the sCD4 concentration in the syringe is 40 μM; the reference cellcontains degassed Milli-Q water. Envelope proteins in the sample cellare titrated to saturation by the stepwise addition of 10 μl of sCD4from the syringe at 400-s intervals at 37° C. The heat evolved upon eachinjection of sCD4 is calculated from the integral of the calorimetricsignal. The heat of dilution of sCD4 is subtracted from the heat ofreaction with gp120 in order to obtain the heat released due to theEnv-sCD4 binding reaction. Molar concentrations of the proteins arecalculated by standard methods, and the values for enthalpy (ΔH),entropy (ΔS), and the association constant (K_(a)) are calculated byfitting the data to a nonlinear least-squares analysis using Originsoftware.

Antigenic Analysis

The antigenicities of uncrosslinked/non-stabilized and dityrosinecrosslinked/stabilized envelope proteins are determined by standardenzyme-linked immunosorbent assay (ELISA) using a panel ofnon-neutralizing, neutralizing, and broadly neutralizing antibodies,including the F105, b12, 2F5, 4E10, D5, 17b (+/−soluble CD4), 15e, D5,b6, PA1, CA13, G3-519, 2G12, and 7B2 antibodies.

Corning high-protein-binding ELISA plates are coated with 400 ng perwell of Galanthus nivalis lectin (catalog no. L8275-5MG; Sigma) in 100μl of PBS (pH 7.4) at 4° C. overnight. The next day, the lectin isremoved, the wells are blocked for 3 hrs at room temperature with PBScontaining 2% fat-free milk and 4% fetal calf serum, and the wells arewashes five times with wash buffer (PBS with 0.2% Tween 20).Subsequently, the wells are incubated with 200 ng of crosslinked anduncrosslinked Env protein in 100 μl of PBS for 2 hrs at roomtemperature, followed by five washes and incubation with 100 μl ofdifferent monoclonal antibody solutions that are fivefold seriallydiluted starting with 20 μg/ml of the initial concentration in dilutionbuffer (1:10-diluted blocking buffer), and incubated for 1 hr at roomtemperature. The wells then are washed and incubated for 1 hr at roomtemperature with 100 μl of a horseradish peroxidase (HRP)-conjugatedanti-human IgG (catalog no. 109-036-097; Jackson ImmunoResearchLaboratories, Inc.) solution at a 1:10,000 dilution in antibody dilutionbuffer. After five subsequent washes, 100 μl of the colorimetricperoxide enzyme immunoassay substrate (3,3′,5,5′-tetramethylbenzidine;Bio-Rad) is added to each well, and the reaction is stopped by adding100 μl of 1M sulfuric acid to the mixture. The OD of the wells is readat 450 nm using an ELISA plate reader. All samples are run in duplicate.The average OD of negative control wells containing bovine serum albumin(BSA) is subtracted from the average OD of experimental wells to obtainfinal OD values.

Immunization and Characterization of Immune Sera Immunization

New Zealand White rabbits (approximately 12 week old females) areinoculated by intradermal injection with 125 μg of proteins emulsifiedin a 1:1 dilution of Ribi adjuvant (Corixa, Hamilton, Mont.) in a totalvolume of 1 ml. One inoculation of 500 μl each is administered in eachhind leg within 2 hrs of preparation. Boosting inoculations are injectedat 4-week intervals. Test bleeds are collected 10 days after eachbooster inoculation. Blood is incubated at room temperature for 2 hrsfor clotting and centrifugated for 10 min at 2,000×g, and the clottedcomponents are discarded. The serum is heat inactivated at 56° C. for 1hr and stored at −20° C. for subsequent analysis.

Characterization of Immunized Sera

To determine the anti-gp 140 antibody titers in immunized sera, ELISAassays are preformed, essentially as described above. Plates are coatedwith 200 ng of wild-type gp120 and gp41 monomeric, dimeric and trimericspike complex Env protein in 100 μl of PBS per well. After blocking andwashes, fivefold serial dilutions (starting at 1/200) of the sera fromimmunized rabbits are added in duplicate wells and incubated for 2 hrsat room temperature. Following washes, the wells are incubated with a1:10,000 dilution of HRP-conjugated anti-rabbit IgG (catalog no.111-035-046; Jackson ImmunoResearch Laboratories, Inc.) and developedwith HRP substrate, and the ODs are read at 450 nm.

Neutralization and Virus Entry Assays Pseudotyped Virus Preparation

HIV-1 is pseudotyped with selected envelope glycoproteins bycotransfection of an env expression vector and viral genomic DNA with adeletion of Env into 293T cells. Following the production of pseudotypedvirus, a luciferase-based neutralization assay is performed aspreviously described in detail in Li et al. 2006 (Li et al., 2006.Characterization of antibody responses elicited by humanimmunodeficiency virus type 1 primary isolate trimeric and monomericenvelope glycoproteins in selected adjuvants. J Virol 80: 1414-1426) andLi et al., 2005 (Li et al., 2005 Human immunodeficiency virus type 1 envclones from acute and early subtype B infections for standardizedassessments of vaccine-elicited neutralizing antibodies. J Virol 79:10108-10125).

HIV Infection Assay

TZM-bl cells expressing CD4, CXCR4, and CCR5, and containingTat-responsive reporter genes for firefly luciferase and the Escherichiacoli β-galactosidase gene under the regulatory control of the HIV-1 longterminal repeat, are used for HIV-1 infection. The level of HIV-1infection is quantified by measuring relative light units (RLU) ofluminescence, which is directly proportional to the amount of viralinfection. The assays are performed using a 96-well microtiter plateformat with 10,000 TZM-bl cells per well. This HIV infection assay isdescribed in detail in Li et al., 2005 (Li et al., 2005 Humanimmunodeficiency virus type 1 env clones from acute and early subtype Binfections for standardized assessments of vaccine-elicited neutralizingantibodies. J Virol 79: 10108-10125).

Neutralization Assays

For neutralization assays, each pseudotyped virus stock is diluted to alevel that produced approximately 100,000 to 500,000 RLU. The percentageof virus neutralization by each immune serum sample is derived bycalculating the reduction in RLUs in the test wells compared to the RLUsin the wells containing pre-immune serum from the corresponding animal.To control for nonspecific neutralization in protein-immunized rabbits,sera from two animals immunized with BSA are analyzed. All serum samplesare also assayed for neutralizing activity against a pseudovirusexpressing the amphotropic murine leukemia virus envelope to test fornon-HIV-1-specific plasma effects. Neutralization of HIV-2 strain7312A/V434M is performed as described in Decker et al., 2005 (Decker etal., 2005. Antigenic Conservation and immunogenicity of the HIVcorecptor binding site. J Exp Med 201: 1407-1419). Pseudovirus stock istreated with mock media or with 0.5 μg/ml of sCD4 (50% inhibitoryconcentration [IC50] for entry of this virus) for 1 hr before addingsera. The remainder of the assay is done as described above. Tocalculate the percent neutralization with sCD4 present in the assay, thebaseline RLU is the value measured with virus plus sCD4 and no serum. Toobtain IC50 data, fivefold serial dilutions of immune sera are incubatedwith viruses before infection of target cells. Antiserum dose-responsecurves are fit with a nonlinear function, and the IC50 for thecorresponding virus is calculated by a least-squares regressionanalysis. Statistical analysis of the IC50 titers is performed with theunpaired t test (GraphPad Prism software package 3.0; GraphPad SoftwareInc., San Diego, Calif.).

Virus Entry Assays

WT and mutant pseudotyped YU2 viruses are produced by cotransfection ofenvelope glycoprotein expressor plasmids and viral genomic DNA with adeletion of the env gene into 293T cells, as described above.Pseudovirus titers are adjusted by p24 ELISA (Beckman Coulter) accordingto the manufacturer's protocol. Equivalent doses of virus suspended in a40 μl volume are then mixed with 20 μl of TZM-bl cells (10,000 cells)and 10 μl of medium on 96-well plates and incubated overnight at 37° C.The following day, 130 μl of cell culture medium is added to each welland incubated for an additional 24 hrs. Cell culture medium then isremoved from all wells, and 50 μl of cell lysis buffer (Promega,Madison, Wis.) is added. Thirty microliters of cell lysis supernatant istransferred onto a new plate containing substrate for the measurement ofluminescence using a luminometer. The RLU produced by the wells aremeasured and used to calculate viral entry. To determineantibody-mediated neutralization of HIV-1 entry, each viral inoculum ispreincubated with fourfold serial dilutions of antibody in 50 μl ofmedium for 1 h at 37° C. After virus-antibody incubation, the TZM-bltarget cells are added to the wells

Example 2

The most potent broadly neutralizing antibodies to HIV bind Envtrimer-specific quaternary neutralizing epitopes (QNEs), but the Envtrimer is too unstable to maintain its quaternary structure and presenttheses QNEs. In preliminary studies using a recombinant, soluble HIV Envtrimer, we have demonstrated that we can use dityrosine (DT)crosslinking to conformationally lock the Env immunogen in its native,trimeric conformation, so that it improves binding to the most potentHIV quaternary broadly neutralizing antibodies. Antibody responses tothese epitopes have the potential to be protective against the enormousbreadth of HIV strains and clades in circulation. By applying targetedDT “staples” to covalently cross-link the trimerizing interactions atthe apex of the native spike, we have successfully engineeredconformationally locked, soluble Env trimmers with fully preserved QNEs.

The HIV envelope spike is trimerized through well characterized,interactions at its base as well as interactions at the spike's apex. Inorder to stabilize the trimerizing interactions at the apex of thespike, we introduced tyrosine substitutions to generate engineered HIVspike proteins, and then expressed, purified, and DT cross-linked theengineered proteins. FIG. 1 shows the results of an analysis of DTcross-linked Env gp140 trimers. DTspecific spectrofluorometry identifiedand quantified DT crosslinks in the HIV Env gp140 variant with tyrosinesubstitution in V1/V2 before and after DT cross-linking Coomassiestaining and a-HIV Env Western blot of purified gp 140 trimer are alsoshown in FIG. 1. These confirmed the presence of intermolecularcross-linking.

By fluorescence, we identified seven variants that formedintermolecular, trimerizing cross-links with an average of80%+efficiency prior to any optimization, as quantified usingDT-specific excitation (320 nm) and emission (405 nm) wavelengths. Weassayed the ability of these constructs to bind conformational andtrimer-specific broadly neutralizing antibodies. DT crosslinking fullypreserved binding of the anti-CD4 binding site on the broadlyneutralizing antibody b12 (which binds both protomers and trimers) andthe anti-V2 broadly neutralizing antibody PG9 (which preferentiallybinds trimers, but also binds monomers). In addition, conformationallocking also significantly reduced binding to non-neutralizingmonoclonal antibodies (such as b6 and b13) in ELISA assays (data notshown). The position of the DT bonds was confirmed by tandem massspectrometry (MS/MS) of tryptic fragments of the DT-Env trimer.Importantly, we found that a conformationally locked HIV Env trimerbinds significantly better to one of the most extremely broadlyneutralizing and potent anti-HIV Env broadly neutralizing antibodies,PG16, by comparison to the wild type protomer. FIG. 2 provides theresults of an ELISA assay. The lower line represents binding of a wildtype (WT) HIV Env protomer to PG16, while the upper line representsbinding of a conformationally locked trimer to PG16. The PG16 epitope isonly presented on the native/functional HIV envelope trimer. ImprovedPG16 binding correlated with a significant reduction in binding to apoorly neutralizing anti-V2 monoclonal antibody, CH58 (data not shown),that binds an α-helical conformer of an overlapping epitope that PG16binds as a β-sheet. The “DT-locked” soluble HIV Env trimer can be testedin various assays to assess its immunogenicity in animals and othercharacteristics. Suitable assays include those described in the previousExamples, those described elsewhere in the specification, those known inthe art.

The invention as described herein is not to be limited in scope to thespecific embodiments and Examples provided, which are intended toprovide illustrations of several aspects of the invention. Variousmodifications of the specific embodiments and examples described herewill be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the present invention.

A number of references are cited herein. For the purposes ofjurisdictions which allow incorporation by reference only, the entiredisclosures of each of the references cited herein are incorporated byreference in their entireties.

The present invention may also be further described and defined in termsof the following claims.

1. A method for producing an immunogen, the method comprising: (a)obtaining a viral protein or protein complex in one or moreconformations that favor the elicitation of protective immune responses,(b) identifying one or more regions in the tertiary and/or quaternarystructure of the viral protein or protein complex in which theintroduction of one or more cross-links could stabilize the conformationof an antibody epitope, (c) introducing into the viral protein orprotein complex one or more dityrosine cross-links at one or more of theregions identified in step (b) to form an engineered viral protein orprotein complex, wherein the engineered viral protein or protein complexhas one or more properties selected from the group consisting of: i.enhanced ability bind to a neutralizing antibody as compared to theviral protein or protein complex, ii. enhanced ability bind to a broadlyneutralizing antibody as compared to the viral protein or proteincomplex, iii. enhanced ability bind to and activate B cell receptors ascompared to the viral protein or protein complex, iv. enhanced abilityto elicit an antibody response in an animal as compared to the viralprotein or protein complex, v. enhanced ability to elicit a protectiveantibody response in an animal as compared to the viral protein orprotein complex, vi. enhanced ability to elicit production ofneutralizing antibodies in an animal as compared to the viral protein orprotein complex, vii. enhanced ability to elicit production of broadlyneutralizing antibodies in an animal as compared to the viral protein orprotein complex, viii. enhanced ability to elicit a protective immuneresponse in an animal as compared to the viral protein or proteincomplex, and ix. enhanced ability to bind to and elicit production ofantibodies that recognize quaternary neutralizing epitopes in an animalas compared to the viral protein or protein complex.
 2. The method ofclaim 1, further comprising performing an assay to assess the ability ofthe engineered viral protein or protein complex to bind to aneutralizing antibody, bind to a broadly neutralizing antibody, bind toand activate B cell receptors, elicit an antibody response in an animal,elicit a protective antibody response in an animal, elicit production ofneutralizing antibodies in an animal, elicit production of broadlyneutralizing antibodies in an animal, elicit a protective immuneresponse in an animal, and/or elicit production of antibodies thatrecognize quaternary neutralizing epitopes in an animal.
 3. The methodof claim 1, wherein at least one dityrosine cross-link originates from apoint mutation to tyrosine.
 4. The method of claim 1, further comprisingintroducing into the viral protein or protein complex one or more pointmutations to tyrosine at one or more of the regions identified in step(b), and then subsequently introducing one or more dityrosinecross-links to form the engineered viral protein or protein complex. 5.The method of claim 1, wherein the engineered viral protein or proteincomplex is useful as a vaccine immunogen in a mammalian subject.
 6. Themethod of claim 1, wherein the engineered viral protein or proteincomplex is useful as a vaccine immunogen in a human subject.
 7. Themethod of claim 1, wherein the viral protein or protein complex isderived from a virus from the group consisting of Herpesvirales,Ligamenvirales, Mononegavirales, Nidovirales, Picornavirales,Lentiviruses, Human Immunodeficiency Viruses, Retroviruses,Orthomyxoviruses, Paramyxovirus, Influenza viruses, Poxviruses,Flaviviruses, Togaviruses, Coronaviruses, Rhabdoviruses, Bunyaviruses,Filoviruses, Reoviruses, Mononegavirales, Hepadnaviruses, and Hepatitisviruses.
 8. The method of claim 1, wherein the viral protein or proteincomplex is a viral envelope protein or protein complex.
 9. The method ofclaim 1, wherein the engineered viral protein or protein complex issoluble and does not form aggregates during production or when stored ata high concentration.
 10. A pharmaceutical composition comprising: apharmaceutically effective amount of an engineered viral protein orprotein complex made using the method of claim 1, and a pharmaceuticallyacceptable carrier.
 11. A method for producing an immunogen, the methodcomprising: (a) obtaining a viral protein or protein complex in one ormore conformations that favor the elicitation of protective immuneresponses, (b) introducing into the viral protein or protein complex oneor more cross-links that are stable under physiological conditions,wherein the engineered viral protein or protein complex has one or moreof properties selected from the group consisting of: i. enhanced abilitybind to a neutralizing antibody as compared to the viral protein orprotein complex, ii. enhanced ability bind to a broadly neutralizingantibody as compared to the viral protein or protein complex, iii.enhanced ability bind to and activate B cell receptors as compared tothe viral protein or protein complex, iv. enhanced ability to elicit anantibody response in an animal as compared to the viral protein orprotein complex, v. enhanced ability to elicit a protective antibodyresponse in an animal as compared to the viral protein or proteincomplex, vi. enhanced ability to elicit production of neutralizingantibodies in an animal as compared to the viral protein or proteincomplex, vii. enhanced ability to elicit production of broadlyneutralizing antibodies in an animal as compared to the viral protein orprotein complex, viii. enhanced ability to elicit a protective immuneresponse in an animal as compared to the viral protein or proteincomplex, and ix. enhanced ability to bind to and elicit production ofantibodies that recognize quaternary neutralizing epitopes in an animalas compared to the viral protein or protein complex.
 12. The method ofclaim 11, further comprising performing an assay to assess the abilityof the engineered viral protein or protein complex to bind to aneutralizing antibody, bind to a broadly neutralizing antibody, bind toand activate B cell receptors, elicit an antibody response in an animal,elicit a protective antibody response in an animal, elicit production ofneutralizing antibodies in an animal, elicit production of broadlyneutralizing antibodies in an animal, elicit a protective immuneresponse in an animal, and/or elicit production of antibodies thatrecognize quaternary neutralizing epitopes in an animal.
 13. The methodof claim 11, wherein the cross-link is targeted to identified andselected positions within the protein or protein complex's tertiary orquaternary structure.
 14. The method of claim 11, wherein thecross-links comprise dityrosine cross links.
 15. The method of claim 13,wherein at least one dityrosine cross-link originates from a pointmutation to tyrosine.
 16. The method of claim 13, further comprisingintroducing into the viral protein or protein complex one or more pointmutations to tyrosine at one or more of the positions identified in thetertiary and/or quaternary structure of the viral protein or proteincomplex such that the introduction of one or more cross-links couldstabilize the conformation of an antibody epitope, and then subsequentlyintroducing one or more dityrosine cross-links to form the engineeredviral protein or protein complex.
 17. The method of claim 11, whereinthe engineered viral protein or protein complex is useful as a vaccineimmunogen in a mammalian subject.
 18. The method of claim 11, whereinthe engineered viral protein or protein complex is useful as a vaccineimmunogen in a human subject.
 19. The method of claim 11, wherein theviral protein or protein complex is derived from a virus belonging thegroup consisting of Herpesvirales, Ligamenvirales, Mononegavirales,Nidovirales, Picornavirales, Lentiviruses, Human ImmunodeficiencyViruses, Retroviruses, Orthomyxoviruses, Paramyxovirus, Influenzaviruses, Poxviruses, Flaviviruses, Togaviruses, Coronaviruses,Rhabdoviruses, Bunyaviruses, Filoviruses, Reoviruses, Mononegavirales,Hepadnaviruses, and Hepatitis viruses.
 20. The method of claim 11,wherein the viral protein or protein complex is a viral envelope proteinor protein complex.
 21. The method of claim 11, wherein the engineeredviral protein or protein complex is soluble and does not form aggregatesduring production or when stored at a high concentration.
 22. Apharmaceutical composition comprising: a pharmaceutically effectiveamount of an engineered viral protein or protein complex made using themethod of claim 11, and a pharmaceutically acceptable carrier.